Sample records for klebsiella pneumoniae hydrazine

The bacterial pathogen Klebsiellapneumoniae is a cause of community- and hospital-acquired lung, urinary tract, and blood stream infections. A common contaminant of indwelling catheters, it is theorized that a common infection pathway for this organism is via shedding of aggregates off of biofilm colonies. In an effort to better understand bacterial proliferation in the host bloodstream, we develop a PDE model for the flocculation dynamics of Klebsiellapneumoniae in suspension. Existence and uniqueness results are provided, as well as a brief description of the numerical approximation scheme. We generate artificial data and illustrate the requirements to accurately identify proliferation, aggregation, and fragmentation of flocs in the experimental domain of interest. PMID:18071828

K. pneumoniae can present as two forms of community-acquired pneumonia, acute and chronic. Although acute pneumonia may turn into necrotizing pneumonia, which results in a prolonged clinical course, it often has a rapidly progressive clinical course. In contrast, chronic Klebsiellapneumonia runs a protracted indolent course that mimics other chronic pulmonary infections and malignancies. Herein, we present two cases of chronic Klebsiellapneumonia. The diagnosis was made by microorganism identification, as well as absence of other potential causes. Clinical and radiographic findings improved after a prolonged course of antibiotic therapy. PMID:26543615

A new hypervirulent (hypermucoviscous) variant of Klebsiellapneumoniae has emerged. First described in the Asian Pacific Rim, it now increasingly recognized in Western countries. Defining clinical features are the ability to cause serious, life-threatening community-acquired infection in younger healthy hosts, including liver abscess, pneumonia, meningitis and endophthalmitis and the ability to metastatically spread, an unusual feature for enteric Gram-negative bacilli in the non-immunocompromised. Despite infecting a healthier population, significant morbidity and mortality occurs. Although epidemiologic features are still being defined, colonization, particularly intestinal colonization, appears to be a critical step leading to infection. However the route of entry remains unclear. The majority of cases described to date are in Asians, raising the issue of a genetic predisposition vs. geospecific strain acquisition. The traits that enhance its virulence when compared with “classical” K. pneumoniae are the ability to more efficiently acquire iron and perhaps an increase in capsule production, which confers the hypermucoviscous phenotype. An objective diagnostic test suitable for routine use in the clinical microbiology laboratory is needed. If/when these strains become increasingly resistant to antimicrobials, we will be faced with a frightening clinical scenario. PMID:23302790

The continuing emergence of infections due to multidrug resistant bacteria is a serious public health problem. Klebsiellapneumoniae, which commonly acquires resistance encoded on mobile genetic elements, including ones that encode carbapenemases, is a prime example. K. pneumoniae carrying such genetic material, including both blaKPC and genes encoding metallo-β-lactamases, have spread globally. Many carbapenemase-producing K. pneumoniae are resistant to multiple antibiotic classes beyond β-lactams, including tetracyclines, aminoglycosides, and fluoroquinolones. The optimal treatment, if any, for infections due to these organisms is unclear but, paradoxically, appears to often require the inclusion of an optimally administered carbapenem. PMID:25343037

A biological inoculant for enhancing the growth of plants is disclosed. The inoculant includes the bacterial strains Herbaspirillum seropedicae 2A, Pantoea agglomerans P101, Pantoea agglomerans P102, Klebsiellapneumoniae 342, Klebsiellapneumoniae zmvsy, Herbaspirillum seropedicae Z152, Gluconacetobacter diazotrophicus PA15, with or without a carrier. The inoculant also includes strains of the bacterium Pantoea agglomerans and K. pneumoniae which are able to enhance the growth of cereal grasses. Also disclosed are the novel bacterial strains Herbaspirillum seropedicae 2A, Pantoea agglomerans P101 and P102, and Klebsiellapneumoniae 342 and zmvsy.

Introduction. Klebsiellapneumoniae is a well-known cause of liver abscess. Higher rates of liver abscess associated with Klebsiellapneumoniae are seen in Taiwan. Metastatic endophthalmitis is a common complication associated with a poor prognosis despite aggressive therapy. Case Report. We report a case of a 67-year-old Korean female with Klebsiellapneumoniae liver abscess. The patient developed metastatic endophthalmitis and ultimately succumbed to her disease despite aggressive medical and surgical treatment. Conclusion. Dissemination of Klebsiellapneumoniae is associated with significant morbidity and mortality. Liver abscesses preferably should be treated with percutaneous drainage, but surgical treatment is needed in some cases. Metastatic spread to the eye is a common complication that must be treated aggressively with intravenous antibiotics and surgical intervention if necessary. PMID:26788530

Fecal coliform-positive, capsule-forming Klebsiellapneumoniae cells were observed in high densities (10(4) to 10(8) CFU/100 ml) in two commercial batches of frozen orange juice concentrate at a cannery in Puerto Rico. Contamination of both lots was gross and included off colors and odors. Isolates of K. pneumoniae from these concentrates revealed growth at 4, 25, and 34 degrees C with generation times from 0.39 to 1.84 h. PMID:3893321

Abstract Klebsiella species are a common cause of community- and nosocomial-acquired pneumonia. Antibiotic resistance to the class of carbapenem in patients with pneumonia caused by Klebsiella species is unusual. New studies report carbapenem resistance in patients with pneumonia caused by Klebsiella species. This article examines, retrospectively, antibiotic resistance in patients with community- and nosocomial-acquired pneumonia caused by Klebsiella species. The data of all patients with community- and nosocomial-acquired pneumonia caused by Klebsiella species were collected from the hospital charts at the HELIOS Clinic, Witten/Herdecke University, Wuppertal, Germany, within the study period 2004 to 2014. An antibiogram was created from all of the study patients with pneumonia caused by Klebsiella species. Sensitivity and resistance profiles were performed for the different antibiotics that have been consistently used in the treatment of patients with pneumonia caused by Klebsiella species. All demographic, clinical, and laboratory data of all of the patients with pneumonia caused by Klebsiella species were collected from the patients’ records. During the study period of January 1, 2004, to August 12, 2014, 149 patients were identified with community- and nosocomial-acquired pneumonia affected by Klebsiella species. These patients had a mean age of 70.6 ± 13 (107 [71.8%, 95% CI 64.6%–79%] men and 42 [28.2%, 95% CI 21%–35.4%] women). In all of the patients with pneumonia caused by Klebsiella species, there was resistance to ampicillin (P pneumonia caused by Klebsiella species (75.3%) also showed resistance to piperacillin (P pneumonia caused by Klebsiella species showed resistance to imipenem or meropenem (P pneumonia caused by Klebsiella species. PMID:25674753

Klebsiella species are a common cause of community- and nosocomial-acquired pneumonia. Antibiotic resistance to the class of carbapenem in patients with pneumonia caused by Klebsiella species is unusual. New studies report carbapenem resistance in patients with pneumonia caused by Klebsiella species.This article examines, retrospectively, antibiotic resistance in patients with community- and nosocomial-acquired pneumonia caused by Klebsiella species.The data of all patients with community- and nosocomial-acquired pneumonia caused by Klebsiella species were collected from the hospital charts at the HELIOS Clinic, Witten/Herdecke University, Wuppertal, Germany, within the study period 2004 to 2014. An antibiogram was created from all of the study patients with pneumonia caused by Klebsiella species. Sensitivity and resistance profiles were performed for the different antibiotics that have been consistently used in the treatment of patients with pneumonia caused by Klebsiella species. All demographic, clinical, and laboratory data of all of the patients with pneumonia caused by Klebsiella species were collected from the patients' records.During the study period of January 1, 2004, to August 12, 2014, 149 patients were identified with community- and nosocomial-acquired pneumonia affected by Klebsiella species. These patients had a mean age of 70.6 ± 13 (107 [71.8%, 95% CI 64.6%-79%] men and 42 [28.2%, 95% CI 21%-35.4%] women). In all of the patients with pneumonia caused by Klebsiella species, there was resistance to ampicillin (P pneumonia caused by Klebsiella species (75.3%) also showed resistance to piperacillin (P pneumonia caused by Klebsiella species showed resistance to imipenem or meropenem (P pneumonia caused by Klebsiella species. PMID:25674753

The current emergence of antibiotic-resistant bacteria causes major problems in hospitals worldwide. To survive within the host, bacterial pathogens exploit several escape mechanisms to prevent detection and killing by the immune system. As a major player in immune defense, the complement system recognizes and destroys bacteria via different effector mechanisms. The complement system can label bacteria for phagocytosis or directly kill Gram-negative bacteria via insertion of a pore-forming complex in the bacterial membrane. The multi-drug resistant pathogen Klebsiellapneumoniae exploits several mechanisms to resist complement. In this review, we present an overview of strategies used by K. pneumoniae to prevent recognition and killing by the complement system. Understanding these complement evasion strategies is crucial for the development of innovative strategies to combat K. pneumoniae. PMID:27364766

The ability of pathogenic bacteria to aggregate and form biofilm represents a great problem for public health, since they present extracellular components that encase these micro-organisms, making them more resistant to antibiotics and host immune attack. This may become worse when antibiotic-resistant bacterial strains form biofilms. However, antibiofilm screens with different compounds may reveal potential therapies to prevent/treat biofilm infections. Here, we focused on Klebsiellapneumoniae, an opportunistic bacterium that causes different types of infections, including in the bloodstream, meninges, lungs, urinary system and at surgical sites. We also highlight aspects involved in the formation and maintenance of K. pneumoniae biofilms, as well as resistance and the emergence of new trends to combat this health challenge. PMID:27064296

Attempts at improving chemical fixation for electron-microscopic observation of the capsule of Klebsiellapneumoniae were made. The capsule was preserved by using alcian blue - lanthanum and tris-(1-aziridinyl) phosphine oxide (TAPO) - aldehyde - osmium procedures. Despite the different retention of the overall capsular material and minor variations in morphological details, in both cases the interpretation of ultrastructural patterns suggested that the capsule be composed of a meshed network of thin polysaccharide fibrils radiating from the cell wall. This organization is in keeping with all recognized chemical properties of bacterial polysaccharide capsules or, at least, does not contradict them. Moreover, an effective preservation of bacterial structures other than capsule has been obtained, mostly in specimens fixed by the TAPO-aldehyde-osmium method, a fact which gives further reliability to the technical approach used for capsule visualization. PMID:326360

ABSTRACT Highly invasive, community-acquired Klebsiellapneumoniae infections have recently emerged, resulting in pyogenic liver abscesses. These infections are caused by hypervirulent K. pneumoniae (hvKP) isolates primarily of capsule serotype K1 or K2. Hypervirulent K1 isolates belong to clonal complex 23 (CC23), indicating that this clonal lineage has a specific genetic background conferring hypervirulence. Here, we apply whole-genome sequencing to a collection of K. pneumoniae isolates to characterize the phylogenetic background of hvKP isolates with an emphasis on CC23. Most of the hvKP isolates belonged to CC23 and grouped into a distinct monophyletic clade, revealing that CC23 is a unique clonal lineage, clearly distinct from nonhypervirulent strains. Separate phylogenetic analyses of the CC23 isolates indicated that the CC23 lineage evolved recently by clonal expansion from a single common ancestor. Limited grouping according to geographical origin was observed, suggesting that CC23 has spread globally through multiple international transmissions. Conversely, hypervirulent K2 strains clustered in genetically unrelated groups. Strikingly, homologues of a large virulence plasmid were detected in all hvKP clonal lineages, indicating a key role in K. pneumoniae hypervirulence. The plasmid encodes two siderophores, aerobactin and salmochelin, and RmpA (regulator of the mucoid phenotype); all these factors were found to be restricted to hvKP isolates. Genomic comparisons revealed additional factors specifically associated with CC23. These included a distinct variant of a genomic island encoding yersiniabactin, colibactin, and microcin E492. Furthermore, additional novel genomic regions unique to CC23 were revealed which may also be involved in the increased virulence of this important clonal lineage. PMID:26199326

Klebsiellapneumoniae is a Gram-negative pathogen frequently associated with antibiotic-resistant nosocomial infections. Bacteriophage therapy against K. pneumoniae may be possible to combat these infections. The following describes the complete genome sequence and key features of the pseudo-T-even K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae myophage Miro. PMID:26430050

Klebsiellapneumoniae is a Gram-negative pathogen frequently associated with antibiotic-resistant nosocomial infections. Bacteriophage therapy against K. pneumoniae may be possible to combat these infections. The following describes the complete genome sequence and key features of the pseudo-T-even K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae myophage Miro. PMID:26430050

Klebsiellapneumoniae is a nosocomial pathogen of emerging importance and displays resistance to broad-spectrum antibiotics, such as carbapenems. Here, we report the genome sequences of five clinical K. pneumoniae isolates, four of which are carbapenem resistant. Carbapenem resistance is conferred by hydrolyzing class A β-lactamases found adjacent to transposases. PMID:26966211

Klebsiellapneumoniae is a Gram-negative bacterium in the family Enterobacteriaceae. It is associated with numerous nosocomial infections, including respiratory and urinary tract infections in humans. The following reports the complete genome sequence of K. pneumoniae carbapenemase-producing K. pneumoniae T1-like siphophage Sushi and describes its major features. PMID:26337889

Klebsiellapneumoniae is a Gram-negative bacterium in the family Enterobacteriaceae. It is associated with numerous nosocomial infections, including respiratory and urinary tract infections in humans. The following reports the complete genome sequence of K. pneumoniae carbapenemase-producing K. pneumoniae T1-like siphophage Sushi and describes its major features. PMID:26337889

Klebsiella species is the second most commonly isolated gram-negative organism in sepsis and a frequent causative pathogen in pneumonia. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory responses. We here aimed to investigate the role of RAGE in the host response to Klebsiella (K.) pneumoniaepneumonia and intransally inoculated rage gene deficient (RAGE-/-) and normal wild-type (Wt) mice with K. pneumoniae. Klebsiellapneumonia resulted in an increased pulmonary expression of RAGE. Furthermore, the high-affinity RAGE ligand high mobility group box-1 was upregulated during K. pneumoniaepneumonia. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE-/- mice. RAGE-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. Relative to Wt mice, RAGE-/- mice demonstrated similar lung inflammation, and slightly elevated-if any-cytokine and chemokine levels and unchanged hepatocellular injury. In addition, RAGE-/- mice displayed an unaltered response to intranasally instilled Klebsiella lipopolysaccharide (LPS) with respect to pulmonary cell recruitment and local release of cytokines and chemokines. These data suggest that (endogenous) RAGE protects against K. pneumoniaepneumonia. Also, they demonstrate that RAGE contributes to an effective antibacterial defense during K. pneumoniaepneumonia, at least partly via its participation in the phagocytic properties of professional granulocytes. Additionally, our results indicate that RAGE is not essential for the induction of a local and systemic inflammatory response to either intact Klebsiella or Klebsiella LPS. PMID:26824892

Klebsiella species is the second most commonly isolated gram-negative organism in sepsis and a frequent causative pathogen in pneumonia. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory responses. We here aimed to investigate the role of RAGE in the host response to Klebsiella (K.) pneumoniaepneumonia and intransally inoculated rage gene deficient (RAGE-/-) and normal wild-type (Wt) mice with K. pneumoniae. Klebsiellapneumonia resulted in an increased pulmonary expression of RAGE. Furthermore, the high-affinity RAGE ligand high mobility group box-1 was upregulated during K. pneumoniaepneumonia. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE-/- mice. RAGE-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. Relative to Wt mice, RAGE-/- mice demonstrated similar lung inflammation, and slightly elevated—if any—cytokine and chemokine levels and unchanged hepatocellular injury. In addition, RAGE-/- mice displayed an unaltered response to intranasally instilled Klebsiella lipopolysaccharide (LPS) with respect to pulmonary cell recruitment and local release of cytokines and chemokines. These data suggest that (endogenous) RAGE protects against K. pneumoniaepneumonia. Also, they demonstrate that RAGE contributes to an effective antibacterial defense during K. pneumoniaepneumonia, at least partly via its participation in the phagocytic properties of professional granulocytes. Additionally, our results indicate that RAGE is not essential for the induction of a local and systemic inflammatory response to either intact Klebsiella or Klebsiella LPS. PMID:26824892

Cerebritis is part of a continuum of brain infection and is difficult to diagnose. Cerebritis caused by Klebsiella in immunocompetent adults without predisposing factors such as neurosurgery or penetrating brain injury has not been reported before. We report a case of Klebsiella cerebritis in an adult patient with a proven extracranial focus of infection. We suggest considering cerebritis as a differential diagnosis for altered level of consciousness in patients of severe sepsis, even if an extracranial source of infection is proven. PMID:19881180

Cerebritis is part of a continuum of brain infection and is difficult to diagnose. Cerebritis caused by Klebsiella in immunocompetent adults without predisposing factors such as neurosurgery or penetrating brain injury has not been reported before. We report a case of Klebsiella cerebritis in an adult patient with a proven extracranial focus of infection. We suggest considering cerebritis as a differential diagnosis for altered level of consciousness in patients of severe sepsis, even if an extracranial source of infection is proven. PMID:19881180

The Gram-negative opportunistic pathogen, Klebsiellapneumoniae, is responsible for causing a spectrum of community-acquired and nosocomial infections and typically infects patients with indwelling medical devices, especially urinary catheters, on which this microorganism is able to grow as a biofilm. The increasingly frequent acquisition of antibiotic resistance by K. pneumoniae strains has given rise to a global spread of this multidrug-resistant pathogen, mostly at the hospital level. This scenario is exacerbated when it is noted that intrinsic resistance to antimicrobial agents dramatically increases when K. pneumoniae strains grow as a biofilm. This review will summarize the findings about the antibiotic resistance related to biofilm formation in K. pneumoniae. PMID:25438022

Klebsiellapneumoniae carbapenemases (KPCs) were first identified in 1996 in the USA. Since then, regional outbreaks of KPC-producing K. pneumoniae have occurred in the USA, and have spread internationally. Dissemination of blaKPC involves both horizontal transfer of blaKPC genes and plasmids, and clonal spread. Of epidemiological significance, the international spread of KPC-producing K. pneumoniae is primarily associated with a single multilocus sequence type (ST), ST258, and its related variants. However, the molecular factors contributing to the success of ST258 largely remain unclear. Here, we review the recent progresses in understanding KPC-producing K. pneumoniae that is contributing to our knowledge of plasmid and genome composition and structure among the KPC epidemic clone, and identify possible factors that influence its epidemiological success. PMID:25304194

A strain of Klebsiellapneumoniae was aerosolized and its survival in air at different relative humidities was studied. Survival was dependent upon relative humidity and aerosols were most stable during storage at a relative humidity of 60%. Mice were exposed to aerosols of K. pneumoniae produced at this humidity and lung samples taken at timed intervals after exposure. Fifteen strains of K. pneumoniae were tested for their ability to colonize mice, but only five were detectable in mouse lungs 7 days after exposure. Three of these strains persisted without an increase in bacterial numbers, regardless of the initial inoculum used. Two strains of K. pneumoniae, designated strains 15 and 16, persisted in a similar manner when used at a low dose; however, when the dose received per lung was increased there was a rapid multiplication of bacteria in the lungs. PMID:1499666

Klebsiellapneumoniae is responsible for a wide range of infections, including urinary tract infections, pneumonia, bacteremia, and liver abscesses. In addition to susceptible clinical isolates involved in nosocomial infections, multidrug-resistant (MDR) and hypervirulent (hvKP) strains have evolved separately in distinct clonal groups. The rapid geographic spread of these isolates is of particular concern. However, we still know little about the virulence of K. pneumoniae except for hvKP, whose secrets are beginning to be revealed. The treatment of K. pneumoniae infections is threatened by the emergence of antimicrobial resistance. The dissemination of resistance is associated with genetic mobile elements, such as plasmids that may also carry virulence determinants. A proficient pathogen should be virulent, resistant to antibiotics, and epidemic. However, the interplay between resistance and virulence is poorly understood. Here, we review current knowledge on the topic. PMID:26718943

Carbapenem-resistant Klebsiellapneumoniae (CRKP) is resistant to almost all antimicrobial agents, is associated with substantial morbidity and mortality, and poses a serious threat to public health. The ongoing worldwide spread of this pathogen emphasizes the need for immediate intervention. This article reviews the global spread and risk factors for CRKP colonization/infection, and provides an overview of the strategy to combat CRKP dissemination. PMID:24353433

Klebsiellapneumoniae mutants were obtained after UV irradiation and negative selection with anticapsular serum. Unencapsulation, rather than expression of a structurally altered capsule, was found in the mutants. The mutant strains showed no alterations in their outer membrane proteins and lipopolysaccharide, and a great similarity with the wild type in the properties tested (serum resistance, antimicrobial sensitivity, and lipopolysaccharide-specific bacteriophage sensitivity), with the exception of a higher cell surface hydrophobicity and resistance to bacteriophage FC3-9.

The extensive use of broad spectrum antibiotics, especially the third generation cephalosporins (C3G), was followed by the emergence of newer plasmid mediated betalactamases called extended spectrum betalactamases (ESBLs). To assess the impact of K. pneumoniae resistant to 3GC in Tunisia, this study was conducted in 3 teaching hospitals. A total of 1110 strains of K pneumoniae was collected. The antibiotics susceptibilities were tested by diffusion method using Mueller-Hinton agar. The quality control was regularly performed. I ESBLs producing solates were detected using the double-disc synergy test. Data analysis was done using the Whonet 4 software. 23.6% K. pneumoniae isolates showed phenotype pattern of ESBLs producers. The double-disc synergy test was positive in 75% of the cases. These isolates were recovered from hospitalized patients in different wards but mainly from pediatrics (23.6%), medicine (23.2%), surgery (22.9%), intensive care units (11%) and neonatology (11%). 54% were isolated from urines, 22% from blood cultures. These isolates remained susceptible to imipenem (100%) and most of them to cefoxitin (96.4%) but all had associated resistance to aminoglycosides, quinolones and trimethoprim-sulfamethoxazole. The prevalence of multidrug resistant K. pneumoniae is high. This resistance can be minimized by the implementation of infection control measures including handwashing and isolation procedures. PMID:12071040

We characterized 53 OXA-48-producing Klebsiellapneumoniae (OXA-48-Kp) isolated between 2011 and 2013 in 21 French hospitals. All the isolates were genotyped using MLST and PFGE and the population structure of the species was determined by a nucleotide-based analysis of the entire K. pneumoniae MLST database. Most of the OXA-48-Kp isolates also produced CTX-M-15 and remained susceptible to imipenem and meropenem. The isolates were distributed into 20 STs, of which five were dominant (ST15, ST101, ST147, ST395 and ST405). All the OXA-48-Kp clustered in the major clade of K. pneumoniae KpI. PMID:24942039

A strain of Klebsiellapneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.

Klebsiellapneumoniae causes a wide range of infections, including pneumonias, urinary tract infections, bacteremias, and liver abscesses. Historically, K. pneumoniae has caused serious infection primarily in immunocompromised individuals, but the recent emergence and spread of hypervirulent strains have broadened the number of people susceptible to infections to include those who are healthy and immunosufficient. Furthermore, K. pneumoniae strains have become increasingly resistant to antibiotics, rendering infection by these strains very challenging to treat. The emergence of hypervirulent and antibiotic-resistant strains has driven a number of recent studies. Work has described the worldwide spread of one drug-resistant strain and a host defense axis, interleukin-17 (IL-17), that is important for controlling infection. Four factors, capsule, lipopolysaccharide, fimbriae, and siderophores, have been well studied and are important for virulence in at least one infection model. Several other factors have been less well characterized but are also important in at least one infection model. However, there is a significant amount of heterogeneity in K. pneumoniae strains, and not every factor plays the same critical role in all virulent Klebsiella strains. Recent studies have identified additional K. pneumoniae virulence factors and led to more insights about factors important for the growth of this pathogen at a variety of tissue sites. Many of these genes encode proteins that function in metabolism and the regulation of transcription. However, much work is left to be done in characterizing these newly discovered factors, understanding how infections differ between healthy and immunocompromised patients, and identifying attractive bacterial or host targets for treating these infections. PMID:27307579

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiellapneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern. PMID:26578797

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiellapneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern. PMID:26578797

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiellapneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage. Images PMID:3888963

Background Klebsiellapneumoniae is a frequent nosocomial pathogen, with the multidrug-resistant (MDR) K. pneumoniae being a major public health concern, frequently causing difficult-to-treat infections worldwide. The aim of this study was to investigate the molecular characterization of clinical MDR Klebsiellapneumoniae isolates. Methods A total of 27 non-duplicate MDR K. pneumoniae isolates with a CTX-CIP-AK resistance pattern were investigated for the prevalence of antimicrobial resistance genes including extended spectrum β-lactamase genes (ESBLs), plasmid-mediated quinolone resistance (PMQR) genes, 16S rRNA methylase (16S-RMTase) genes, and integrons by polymerase chain reaction (PCR) amplification and DNA sequencing. Plasmid replicons were typed by PCR-based replicon typing (PBRT). Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to characterize the strain relatedness. Results All the isolates co-harbored 3 or more resistance determinants. OqxAB, CTX-M-type ESBLs and RmtB were the most frequent determinants, distributed among19 (70.4%),18 (66.7%) and 8 (29.6%) strains. Fourteen isolates harbored class 1 integrons, with orfD-aacA4 being the most frequent gene cassette array. Class 3 integrons were less frequently identified and contained the gene cassette array of blaGES-1-blaOXA-10-aac(6′)-Ib. IncFII replicon was most commonly found in this collection. One cluster was observed with ≥80% similarity among profiles obtained by PFGE, and one sequence type (ST) by MLST, namely ST11, was observed in the cluster. Conclusion K. pneumoniae carbapenemase (KPC)–producing ST11 was the main clone detected. Of particular concern was the high prevalence of multiple resistance determinants, classs I integrons and IncFII plasmid replicon among these MDR strains, which provide advantages for the rapid development of MDR strains. PMID:24884610

Klebsiellapneumoniae produces many economically important chemicals. Using glucose as a carbon source, the main metabolic product in K. pneumoniae is 2,3-butanediol. Gluconic acid is an intermediate of the glucose oxidation pathway. In the current study, a metabolic engineering strategy was used to develop a gluconic acid-producing K. pneumoniae strain. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. Gluconic acid accumulation by K. pneumoniae Δgad was an acid-dependent aerobic process, with accumulation observed at pH 5.5 or lower, and at higher levels of oxygen supplementation. Under all other conditions tested, 2,3-butanediol was the main metabolic product of the process. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by K. pneumoniae Δgad, and the conversion ratio of glucose to gluconic acid reached 1 g/g. The K. pneumoniae Δgad described in this study is the first genetically modified strain used for gluconic acid production, and this optimized method for gluconic acid production may have important industrial applications. Gluconic acid is an intermediate of this glucose oxidation pathway. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by the K. pneumoniae Δgad strain, and the conversion ratio of glucose to gluconic acid reached 1 g/g. PMID:27339313

In Klebsiellapneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer. Images PMID:7007348

Klebsiellapneumoniae carbapenemases (KPCs) were originally identified in the USA in 1996. Since then, these versatile β-lactamases have spread internationally among Gram-negative bacteria, especially K pneumoniae, although their precise epidemiology is diverse across countries and regions. The mortality described among patients infected with organisms positive for KPC is high, perhaps as a result of the limited antibiotic options remaining (often colistin, tigecycline, or aminoglycosides). Triple drug combinations using colistin, tigecycline, and imipenem have recently been associated with improved survival among patients with bacteraemia. In this Review, we summarise the epidemiology of KPCs across continents, and discuss issues around detection, present antibiotic options and those in development, treatment outcome and mortality, and infection control. In view of the limitations of present treatments and the paucity of new drugs in the pipeline, infection control must be our primary defence for now. PMID:23969216

In Klebsiellapneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer. PMID:7007348

Klebsiellapneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

The chromosomal DNA that lies between the lacI and lacZ genes of Klebsiellapneumoniae constitutes a 196-base pair intercistronic region that contains regulatory sequences for both genes. The probable locations of specific regulatory elements for both lacI and lacZ genes were determined by analogy with the corresponding Escherichia coli sequences. A recombinational event in ancestral DNA evidently has inverted the transcriptional direction of lacI in K. pneumoniae relative to the transcriptional direction of lacI in E. coli. One end of the inversion was located within a 19-base pair sequence in the K. pneumoniae regulatory region. Sequences partially homologous to these 19 base pairs were found in two locations on either side of the E. coli lacI gene. The nucleotide sequence of the lac regulatory region in K. pneumoniae exhibits more than one possibility for folded tertiary structures. The spatial relationships of transcriptional binding sites differ in two possible structures. Associations of regulatory and transcriptional proteins with the DNA might affect conformation of the regulatory sequences and, as a consequence, transcription of the lac genes. PMID:3897197

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE), especially Klebsiellapneumoniae carbapenemase (KPC)-producing K. pneumoniae, pose an urgent threat in health facilities in the United States and worldwide. K. pneumoniae isolates classified as sequence type 258 (ST258) by multilocus sequence typing are largely responsible for the global spread of KPC. A recent comparative genome study revealed that ST258 K. pneumoniae strains are two distinct genetic clades; however, the molecular origin of ST258 largely remains unknown, and our understanding of the evolution of the two genetic clades is incomplete. Here we compared the genetic structures and single-nucleotide polymorphism (SNP) distributions in the core genomes of strains from two ST258 clades and other STs (ST11, ST442, and ST42). We identified an ~1.1-Mbp region on ST258 genomes that is homogeneous to that of ST442, while the rest of the ST258 genome resembles that of ST11. Our results suggest ST258 is a hybrid clone—80% of the genome originated from ST11-like strains and 20% from ST442-like strains. Meanwhile, we sequenced an ST42 strain that carries the same K-antigen-encoding capsule polysaccharide biosynthesis gene (cps) region as ST258 clade I strains. Comparison of the cps-harboring regions between the ST42 and ST258 strains (clades I and II) suggests the ST258 clade I strains evolved from a clade II strain as a result of cps region replacement. Our findings unravel the molecular evolution history of ST258 strains, an important first step toward the development of diagnostic, therapeutic, and vaccine strategies to combat infections caused by multidrug-resistant K. pneumoniae. PMID:24961694

We report a rare case of Klebsiellapneumoniae meningitis associated with liver abscess, which was successfully treated with cefotaxime (CTX), one of the third-generation cephalosporins. A 53-year-old man was admitted to Keio University Hospital on June 13, 1988, because of a fever and a headache. On June 3, he suddenly started shivering and his temperature rose to 39 degrees C. He then began to complain of polydipsia, polyuria, and a weight loss of 4 kg a week. On June 11, he developed a severe headache. Four years prior to this incident, he had been diagnosed as having diabetes after a routine medical examination, but had neglected to undergo medical treatment. On admission, laboratory data showed leukocytosis, hyperglycemia (394 mg/dl) and ketonuria (4+). A lumbar puncture yielded cloudy cerebrospinal fluid (CSF) containing 500/3 cells/mm8, of which about 70% were neutrophils. A diagnosis of diabetic ketoacidosis and purulent meningitis was made. A treatment with ampicillin (ABPC) and CTX, (12 g/day, each) was begun. On the third day, cultures of a blood specimen and CSF yielded both K. pneumoniae. The MICs of CTX to K. pneumoniae isolated from blood and CSF were both 0.05 microgram/ml. ABPC was discontinued, gentamicin was administered for 2 days, CTX was continued at the same dosage level and an administration of prednisolone 40 mg daily was begun.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2691713

Infections by Carbapenem-Resistant Enterobacteriaceae (CRE), in particular, carbapenem-resistant Klebsiellapneumoniae (CRKp), are a significant public health challenge worldwide. Resistance to carbapenems in enterobacteriaceae is linked to different mechanisms, including the production of the various types of enzymes like KPC, VIM, IMP, NDM, and OXA-48. Despite several attempts to control the spread of these infections at the local and national level, the epidemiological situation for CRKp had worsened in the last years in the Mediterranean area. The rate and types of CRKp isolates greatly differ in the various Mediterranean countries. KPC-producing K. pneumoniae is diffused particularly in the European countries bordering the Mediterranean Sea and is endemic in Greece and Italy. On the contrary, OXA-48-producing K. pneumoniae is endemic in Turkey and Malta and diffused at inter-regional level particularly in some North African and Middle East countries. The spread of these multiresistant pathogens in the world and the Mediterranean countries has been related to various epidemiological factors including the international transfer of patients coming from endemic areas. PMID:27441063

Klebsiellapneumoniae is a leading cause of nosocomial infections in the United States. Due to the emergence of multidrug-resistant strains, phages targeting K. pneumoniae may be a useful alternative against this pathogen. Here, we announce the complete genome of K. pneumoniae pseudo-T-even myophage Matisse and describe its features. PMID:26430049

Klebsiellapneumoniae is a leading cause of nosocomial infections in the United States. Due to the emergence of multidrug-resistant strains, phages targeting K. pneumoniae may be a useful alternative against this pathogen. Here, we announce the complete genome of K. pneumoniae pseudo-T-even myophage Matisse and describe its features. PMID:26430049

From early this decade, Enterobacteriaceae that produce Klebsiellapneumoniae carbapenemases (KPC) were reported in the USA and subsequently worldwide. These KPC-producing bacteria are predominantly involved in nosocomial and systemic infections; although they are mostly Enterobacteriaceae, they can also be, rarely, Pseudomonas aeruginosa isolates. KPC beta lactamases (KPC-1 to KPC-7) confer decreased susceptibility or resistance to virtually all beta lactams. Carbapenems (imipenem, meropenem, and ertapenem) may thus become inefficient for treating enterobacterial infections with KPC-producing bacteria, which are, in addition, resistant to many other non-beta-lactam molecules, leaving few available therapeutic options. Detection of KPC-producing bacteria may be difficult based on routine antibiotic susceptibility testing. It is therefore crucial to implement efficient infection control measures to limit the spread of these pathogens. PMID:19324295

Hospital infections caused by carbapenem-resistant Klebsiellapneumoniae (CRKP) constitute a worldwide problem associated with high rates of treatment failure and mortality. In Greece, CRKP have emerged in 2002 due to VIM carbapenemase production and later due to KPC, NDM and OXA-48-like carbapenemases that have become endemic. The molecular epidemiology of CRKP strains is dynamic, as antibiotic consumption and worldwide traveling are strongly associated with global spread of CRKP isolates. Lately, porin defects, such as disruption of OmpK35 and production of OmpK36 variant, have also contributed to carbapenem resistance. In the coming years, the high prevalence of CRKP will require intense infection control measures, while novel molecular patterns may appear. To our knowledge, this is the first review analyzing the molecular epidemiology of CRKP strains in Greece. PMID:27206024

Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiellapneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics. PMID:26478520

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiellapneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four blaOXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured blaCTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the blaOXA-48 gene in all four isolates was identical to pKPOXA-48N1, a blaOXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify blaOXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of blaOXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern. PMID:26414183

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiellapneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four blaOXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured blaCTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the blaOXA-48 gene in all four isolates was identical to pKPOXA-48N1, a blaOXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify blaOXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of blaOXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern. PMID:26414183

In vitro production of ammonia through biological means poses a number of challenges. The organisms should be able to accumulate considerable concentrations of ammonia in the medium. Secondly, nonphotosynthetic organisms must be supplied with high-energy substrates to carry out the fixation reaction. Thirdly, the organisms must be kept in a viable state to produce ammonia over long periods of time. In this article, preliminary results on the production of ammonia by a mutant strain of Klebsiellapneumoniae in continuous reactor systems are discussed. Continuous production of ammonia becomes feasible through the immobilization of the whole microbial cells and then through the use of the resulting catalyst system in a flow-through reactor. The rationale for immobilizing microbial cells and the advantages of such an approach over traditional fermentation processes are briefly described as they relate to the microbial production of ammonia. The microbial cells can be immobilized in such a way that their viability is still maintained in the immobilized state. This, in turn, obviates addition of cofactors, which is often an expensive step associated with immobilized multi-enzyme systems. Reconstituted bovine-hide collagen as the carrier matrix for fixing the cells was the carrier of choice for our work on immobilized Klebsiella cells. Polyacrylamide gels were examined as an alternate carrier matrix but results from this were found to be inferior to those collagen immobilized cell system.

Klebsiellapneumoniae is an important cause of healthcare-associated infections worldwide. Selective pressure, the extensive use of antibiotics, and the conjugational transmission of antibiotic resistance genes across bacterial species and genera facilitate the emergence of multidrug-resistant (MDR) K. pneumoniae. Here, we examined the occurrence, phenotypes and genetic features of MDR K. pneumoniae isolated from patients in intensive care units (ICUs) at the First Affiliated Hospital of Xiamen University in Xiamen, China, from January to December 2011. Thirty-eight MDR K. pneumoniae strains were collected. These MDR K. pneumoniae isolates possessed at least seven antibiotic resistance determinants, which contribute to the high-level resistance of these bacteria to aminoglycosides, macrolides, quinolones and β-lactams. Among these isolates, 24 strains were extended-spectrum β-lactamase (ESBL) producers, 2 strains were AmpC producers, and 12 strains were both ESBL and AmpC producers. The 38 MDR isolates also contained class I (28/38) and class II integrons (10/38). All 28 class I-positive isolates contained aacC1, aacC4, orfX, orfX' and aadA1 genes. β-lactam resistance was conferred through bla SHV (22/38), bla TEM (10/38), and bla CTX-M (7/38). The highly conserved bla KPC-2 (37/38) and bla OXA-23(1/38) alleles were responsible for carbapenem resistance, and a gyrAsite mutation (27/38) and the plasmid-mediated qnrB gene (13/38) were responsible for quinolone resistance. Repetitive-sequence-based PCR (REP-PCR) fingerprinting of these MDR strains revealed the presence of five groups and sixteen patterns. The MDR strains from unrelated groups showed different drug resistance patterns; however, some homologous strains also showed different drug resistance profiles. Therefore, REP-PCR-based analyses can provide information to evaluate the epidemic status of nosocomial infection caused by MDR K. pneumoniae; however, this test lacks the power to discriminate some

Klebsiellapneumoniae is an important cause of healthcare-associated infections worldwide. Selective pressure, the extensive use of antibiotics, and the conjugational transmission of antibiotic resistance genes across bacterial species and genera facilitate the emergence of multidrug-resistant (MDR) K. pneumoniae. Here, we examined the occurrence, phenotypes and genetic features of MDR K. pneumoniae isolated from patients in intensive care units (ICUs) at the First Affiliated Hospital of Xiamen University in Xiamen, China, from January to December 2011. Thirty-eight MDR K. pneumoniae strains were collected. These MDR K. pneumoniae isolates possessed at least seven antibiotic resistance determinants, which contribute to the high-level resistance of these bacteria to aminoglycosides, macrolides, quinolones and β-lactams. Among these isolates, 24 strains were extended-spectrum β-lactamase (ESBL) producers, 2 strains were AmpC producers, and 12 strains were both ESBL and AmpC producers. The 38 MDR isolates also contained class I (28/38) and class II integrons (10/38). All 28 class I-positive isolates contained aacC1, aacC4, orfX, orfX’ and aadA1 genes. β-lactam resistance was conferred through bla SHV (22/38), bla TEM (10/38), and bla CTX-M (7/38). The highly conserved bla KPC-2 (37/38) and bla OXA-23(1/38) alleles were responsible for carbapenem resistance, and a gyrAsite mutation (27/38) and the plasmid-mediated qnrB gene (13/38) were responsible for quinolone resistance. Repetitive-sequence-based PCR (REP-PCR) fingerprinting of these MDR strains revealed the presence of five groups and sixteen patterns. The MDR strains from unrelated groups showed different drug resistance patterns; however, some homologous strains also showed different drug resistance profiles. Therefore, REP-PCR-based analyses can provide information to evaluate the epidemic status of nosocomial infection caused by MDR K. pneumoniae; however, this test lacks the power to discriminate some

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiellapneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a c...

Klebsiellapneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6-6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %. PMID:26059458

Some important virulence factors have been elucidated in Klebsiellapneumoniae infections. We investigated the relationship between virulence factors and multilocus sequence types (STs) and assessed the risk factors for bacteremia in patients with pneumonia due to K. pneumoniae. From April 2004 through April 2012, a total of 120 K. pneumoniae isolates from patients with pneumonia (23 with bacteremia and 97 without bacteremia) were collected from 10 medical institutions in Japan. Additionally, 10 strains of K. pneumoniae serotype K2 that were isolated >30 years ago were included in this study. These isolates were characterized using multilocus sequence typing (MLST), and the characteristics of their virulence factors, such as hypermucoviscosity phenotype and RmpA and aerobactin production between patients with and without bacteremia, were examined. MLST analysis was performed on the 120 isolates from patients with pneumonia, and some sequence type groups were defined as genetic lineages (GLs). GL65 was more prevalent among patients with bacteremia (21.7%) than in those without bacteremia (7.2%). The majority of the strains with serotype K2 were classified into GL14 or GL65, and rmpA and the gene for aerobactin were present in all GL65-K2 strains but absent in all GL14-K2 strains. In a multivariate analysis, the independent risk factors for bacteremia included GL65 (adjusted odds ratio [AOR], 9.46; 95% confidence interval [CI], 1.81 to 49.31), as well as neoplastic disease (AOR, 9.94; 95% CI, 2.61 to 37.92), immunosuppression (AOR, 17.85; 95% CI, 1.49 to 214.17), and hypoalbuminemia (AOR, 4.76; 95% CI, 1.29 to 17.61). GL65 was more prevalent among patients with bacteremia and was associated with the virulence factors of K. pneumoniae. PMID:25568434

We describe the detection of the CTX-M-12 β-lactamase from a clinical isolate of Klebsiellapneumoniae in Colombia. Screening of nosocomial Klebsiella spp. and Escherichia coli isolates from a network of teaching hospitals revealed the presence of CTX-M enzymes in multiple cities. This is the first description of CTX-M in Colombia. PMID:14742223

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiellapneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine. PMID:12089029

Klebsiellapneumoniae is a zoonotic, Gram-negative member of the family Enterobacteriaceae and is the causative agent of nosocomial septicemic, pneumonic, and urinary tract infections. Recently, pathogenic strains of K. pneumoniae sharing a hypermucoviscosity (HMV) phenotype have been attributed to ...

Carbapenem resistance in Klebsiellapneumoniae, frequently conferred by the blaKPC gene, is a major public health threat. We sequenced a blaKPC-containing strain of K. pneumoniae belonging to the emergent lineage ST941, in order to better understand the evolution of blaKPC within this species. PMID:26823590

Carbapenem resistance in Klebsiellapneumoniae, frequently conferred by the blaKPC gene, is a major public health threat. We sequenced a blaKPC-containing strain of K. pneumoniae belonging to the emergent lineage ST941, in order to better understand the evolution of blaKPC within this species. PMID:26823590

In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-­glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-­oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiellapneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein. PMID:21795809

Ethyl carbamate (EC) is a carcinogenic substance in many fermented foods. Enzymatic removal of ethyl carbamate from fermented foods is an important way to eliminate its potential health damage to consumers. To study the enzymatic properties of an ethyl carbamate hydrolase (urethanase) from Klebsiellapneumoniae, a strain isolated from murine somach, we purified the enzyme using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The molecular mass of this enzyme was estimated to be 55 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its K(m) was 74 mmol/L when EC was used as the substrate. Moreover, its optimal reaction temperature was 55 degrees C, and the optimum pH was 7.0. The activity was enhanced by ethylene diamine tetraacetic acid (EDTA) and dithiothreitol (DTT), but strongly inhibited by Cu2+ and Zn2+. The enzyme was halophilic and tolerant to low concentration of ethanol. Therefore, it has the potential to remove EC from fermented foods. PMID:25007576

The crystal structure of 2-oxo-3-deoxygalactonate kinase from the De Ley–Doudoroff pathway of galactose metabolism has been determined at 2.1 Å resolution. In most organisms, efficient d-galactose utilization requires the highly conserved Leloir pathway that converts d-galactose to d-glucose 1-phosphate. However, in some bacterial and fungal species alternative routes of d-galactose assimilation have been identified. In the so-called De Ley–Doudoroff pathway, d-galactose is metabolized into pyruvate and d-glyceraldehyde 3-phosphate in five consecutive reactions carried out by specific enzymes. The penultimate step in this pathway involves the phosphorylation of 2-oxo-3-deoxygalactonate to 2-oxo-3-deoxygalactonate 6-phosphate catalyzed by 2-oxo-3-deoxygalactonate kinase, with ATP serving as a phosphoryl-group donor. Here, a crystal structure of 2-oxo-3-deoxygalactonate kinase from Klebsiellapneumoniae determined at 2.1 Å resolution is reported, the first structure of an enzyme from the De Ley–Doudoroff pathway. Structural comparison indicates that the enzyme belongs to the ASKHA (acetate and sugar kinases/hsc70/actin) family of phosphotransferases. The protein is composed of two α/β domains, each of which contains a core common to all family members. Additional elements introduced between conserved structural motifs define the unique features of 2-oxo-3-deoxygalactonate kinase and possibly determine the biological function of the protein.

Encapsulated Klebsiellapneumoniae has emerged as one of the most clinically relevant and more frequently encountered opportunistic pathogens in combat wounds as the result of nosocomial infection. In this report, we show that imipenem displayed potent activity against established K. pneumoniae biofilms under both static and flow conditions in vitro. Using a rabbit ear model, we also demonstrated that imipenem was highly effective against preformed K. pneumoniae biofilms in wounds. PMID:24247132

Besides urinary tract infection (UTI) and pneumonia, increased severe liver abscesses caused by Klebsiellapneumoniae (KP), especially in diabetic patients, have been observed in infections acquired in hospitals. This indicates that different KP strains with higher virulence have emerged in recent years. Our goal was to investigate the infectivity of KP isolates in mice from liver abscess or UTI patients. Mice were injected with streptozotocin to induce diabetes. Male ICR mice were infected with KpU1 (UTI strain CG3 for survival experiment only) and KpL1 (liver abscess strain CG5) by tail-vein injection of 5 x 10(4) colony-forming units (CFU) bacterial suspension. The mice survival rates, cytokine level by enzyme-linked immunosorbent assay (ELISA), and bacterial presence in liver tissue by Giemsa stain were examined. The survival rates for the KpL1-infected animals were 28% and 0% in normal and diabetic groups, respectively, whereas, for the KpU1-infected mice, the rates were 100% and 75% during a 30-day observation. Nonsurviving KpL1-infected mice showed > 10(5) bacteria/ml blood and the bacteria appeared in the liver sinus area and inside liver cells. The KpL1-infected mice showed a tendency to increase the blood interleukin 1beta (IL-1beta) level in both nondiabetic and diabetic groups, whereas the tumor necrosis factor-alpha (TNF-alpha) level was significantly decreased in the KpL1-infected diabetic mice (P = 0.002). In conclusion, the KP strain from liver abscess showed a greater virulence in mice than the KP from UTI and was more virulent in diabetic than in nondiabetic mice. The infection with KP from liver abscess significantly decreased the blood TNF-alpha level in diabetes mellitus (DM) mice and the blood IL-1beta level tended to increase in both infected nondiabetic and diabetic groups. High blood bacterial count and appearance of bacteria in liver sinus and cells usually contribute to death of the animals. PMID:16246903

Klebsiella variicola, a bacterium closely genetically related to Klebsiellapneumoniae, is commonly misidentified as K. pneumoniae by biochemical tests. To distinguish between the two bacteria, phylogenetic analysis of the rpoB gene and the identification of unique genes in both bacterial species by multiplex-polymerase chain reaction (PCR) provide the means to reliably identify and genotype K. variicola. In recent years, K. variicola has been described both as the cause of an intrahospital outbreak in a pediatric hospital, which resulted in sepsis in inpatients, and as a frequent cause of bloodstream infections. In the present study, K. pneumoniae and K. variicola were isolated from a unique patient displaying different antimicrobial susceptibility phenotypes and different genotypes of virulence determinants. Eight clinical isolates were obtained at different time intervals; all during a 5-month period. The isolates were identified as K. pneumoniae by an automated identification system. The clinical (biochemical test) and molecular (multiplex-PCR and rpoB gene) characterization identified imipenem resistance in the first six K. pneumoniae ST258 isolates, which encode the SHV-12 cephalosporinase and KPC-3 carbapenemase genes. The two last remaining isolates corresponded to susceptible K. variicola. The bacterial species showed a specific profile of virulence-associated determinants, specifically the fimA, fimH, and ecpRAB fimbrial-encoding genes identified only in K. pneumoniae isolates. However, the entb (enterobactin), mrkD (fimbrial adhesin), uge (epimerase), ureA (urease), and wabG (transferase) genes were shared between both bacterial species. Recent studies attribute a higher mortality rate to K. variicola than to K. pneumonia. This work highlights the identification of K. pneumoniae and the closely related K. variicola isolated from the same patient. The value of distinguishing between these two bacterial species is in their clinical significance, their

A 53-year-old man presented with fevers, productive cough and decreased appetite. He emigrated from Iraq 4 years ago. Chest x-ray revealed a left lung consolidation. Respiratory cultures and two sets of blood cultures grew out pan-susceptible Klebsiellapneumoniae Liver ultrasound revealed a 6.4-cm complex lesion in the left hepatic lobe. A biopsy of the liver lesion produced bloody purulent aspirate; abscess cultures yielded a highly viscous pan-susceptible K. pneumoniaeKlebsiellapneumoniae liver abscess syndrome is a newly described invasive syndrome due to a hypermucoviscous phenotype associated with serotypes K1 and K2 of Klebsiella. Although it is more commonly endemic to the Asian-Pacific region, it has been increasingly reported as an emerging global disease. We present the first case of this syndrome in a patient of middle-eastern descent. We also present pictorial evidence of the microbe's unique viscous, muculent texture grown on agar. PMID:27016535

The activity of dextranase, lactoferrin, lysozyme, and nisin against biofilms composed of either Klebsiellapneumonia or Escherichia coli was examined using the MBEC Assay™. Mature biofilms were treated and then sonicated to remove the adherent biofilm. This material was quantified using a lumines...

Ceftazidime-avibactam is the first antimicrobial approved by the U.S. FDA for the treatment of carbapenem-resistant Enterobacteriaceae. Avibactam, a non-β-lactam β-lactamase inhibitor, inactivates class A serine carbapenemases, including Klebsiellapneumoniae carbapenemase (KPC). We report a KPC-producing K. pneumoniae isolate resistant to ceftazidime-avibactam (MIC, 32/4 μg/ml) from a patient with no prior treatment with ceftazidime-avibactam. PMID:26195508

In the last two decades, Klebsiellapneumoniae demonstrated some characteristics of acquisition of plasmids coding extended spectrum β-lactamases (ESBL). The review data showed an increase in worldwide prevalence of ESBL and a temporal shift in the prevalence of ESBL types in K. pneumoniae during this last decade. CTX-M-15 seems to be the predominant ESBL type in K. pneumoniae in some parts of the world. The dissemination of several nosocomial CTX-M-15-producing K. pneumoniae clones was reported unlike the worldwide dissemination of a single virulent ST131 CTX-M-15 producing Escherichia coli clone. The diversity of plasmids carrying the bla(CTX-M-15) gene in K. pneumoniae suggested the frequent transfer of this gene between different replicons. The acquisition of the bla(CTX-M-15) gene by K. pneumoniae was probably occurred via horizontal transfer from E. coli. PMID:22008131

Klebsiellapneumoniae strain IIEMP-3, isolated from Indonesian tempeh, is a vitamin B12-producing strain that exhibited a different genetic profile from pathogenic isolates. Here we report the draft genome sequence of strain IIEMP-3, which may provide insights on the nature of fermentation, nutrition, and immunological function of Indonesian tempeh. PMID:26950331

This report describes an outbreak of suppurative peritonitis caused by Klebsiellapneumoniae in an adult female of captive golden-handed tamarin (Saguinus midas midas). Two virulent and multidrug-resistant strains were isolated and classified through MLST as ST60 and ST1263. The microbiological diagnosis works as a support tool for preventive measures. PMID:26620445

Klebsiellapneumoniae T2-1-1 was isolated from the human tongue debris and subjected to whole genome sequencing on HiSeq platform and annotated on RAST. The nucleotide sequence of this genome was deposited into DDBJ/EMBL/GenBank under the accession JAQL00000000. PMID:26981378

Here, we present the 6,155,188-bp draft genome sequence of Klebsiellapneumoniae UCD-JA29, isolated from blood cultures from a patient with sepsis at the University of California, Davis Medical Center in Sacramento, California, USA. PMID:27151785

New antibiotic options are urgently needed for the treatment of carbapenem-resistant Enterobacteriaceae infections. We report a 64-year-old female with prolonged hospitalization following an intestinal transplant who developed refractory bacteremia due to a serine carbapenemase-producing pandrug-resistant isolate of Klebsiellapneumoniae. After failing multiple antimicrobial regimens, the patient was successfully treated. PMID:26386029

Klebsiellapneumoniae are Gram negative opportunistic pathogens producing capsular (K) polysaccharides. Seventy-seven different K antigens have been described and they are the basis for K serotyping. Capsular polysaccharides are important virulence factors and have a relevant role for the structure of biofilm communities. Nevertheless, little information is available on the polysaccharides produced in biofilm matrices by Klebsiella spp. In the present study, a clinical isolate of Klebsiellapneumoniae was grown both on cellulose membranes deposited on agar plates, where it formed an adherent biofilm, and in liquid medium, where it formed floating biofilms (flocs). Extraction and purification of the polysaccharide fraction showed that only one main carbohydrate polymer was present in both adherent biofilms and flocs. Composition and linkage analysis, Smith degradation followed by ESI-MS, 1D and 2D NMR spectroscopy revealed that the polysaccharide belong to the type K24 and has the following structure. PMID:27182661

Klebsiellapneumoniae has been known historically as a causal agent of bacterial pneumonia. More recently, K. pneumoniaerepresentatives have been shown to have a broad ecological distribution and are recognized nitrogen-fixers. Previously, we demonstrated the capacity of K. pneumoniae strain Kp 5-1R...

This study presents the first characterization of carbapenem-non-susceptible Klebsiellapneumoniae isolates by means of a structured six-month survey performed in Romania as part of an Europe-wide investigation. Klebsiellapneumoniae clinical isolates from different anatomical sites were tested for antibiotic susceptibility by phenotypic methods and confirmed by PCR for the presence of four carbapenemase genes. Genome macrorestriction fingerprinting with XbaI was used to analyze the relatedness of carbapenemase-producing Klebsiellapneumoniae isolates collected from eight hospitals. Among 75 non-susceptible isolates, 65 were carbapenemase producers. The most frequently identified genotype was OXA-48 (n = 51 isolates), eight isolates were positive for blaNDM-1 gene, four had the blaKPC-2 gene, whereas two were positive for blaVIM-1. The analysis of PFGE profiles of OXA-48 and NDM-1 producing K. pneumoniae suggests inter-hospitals and regional transmission of epidemic clones. This study presents the first description of K. pneumoniae strains harbouring blaKPC-2 and blaVIM-1 genes in Romania. The results of this study highlight the urgent need for the strengthening of hospital infection control measures in Romania in order to curb the further spread of the antibiotic resistance. PMID:26599338

Klebsiella spp. have become an important cause of clinical mastitis in dairy cows in New York State. We describe the occurrence of two Klebsiella mastitis outbreaks on a single dairy farm. Klebsiella isolates from milk, feces, and environmental sources were compared using random amplified polymorphic DNA (RAPD)-PCR typing. The first mastitis outbreak was caused by a single strain of Klebsiellapneumoniae, RAPD type A, which was detected in milk from eight cows. RAPD type A was also isolated from the rubber liners of milking machine units after milking of infected cows and from bedding in the outbreak pen. Predominance of a single strain could indicate contagious transmission of the organism or exposure of multiple cows to an environmental point source. No new cases with RAPD type A were observed after implementation of intervention measures that targeted the prevention of transmission via the milking machine as well as improvement of environmental hygiene. A second outbreak of Klebsiella mastitis that occurred several weeks later was caused by multiple RAPD types, which rules out contagious transmission and indicates opportunistic infections originating from the environment. The diversity of Klebsiella strains as quantified with Simpson's index of discrimination was significantly higher for isolates from fecal, feed, and water samples than for isolates from milk samples. Several isolates from bedding material that had the phenotypic appearance of Klebsiella spp. were identified as being Raoultella planticola and Raoultella terrigena based on rpoB sequencing. PMID:17928424

Klebsiella spp. have become an important cause of clinical mastitis in dairy cows in New York State. We describe the occurrence of two Klebsiella mastitis outbreaks on a single dairy farm. Klebsiella isolates from milk, feces, and environmental sources were compared using random amplified polymorphic DNA (RAPD)-PCR typing. The first mastitis outbreak was caused by a single strain of Klebsiellapneumoniae, RAPD type A, which was detected in milk from eight cows. RAPD type A was also isolated from the rubber liners of milking machine units after milking of infected cows and from bedding in the outbreak pen. Predominance of a single strain could indicate contagious transmission of the organism or exposure of multiple cows to an environmental point source. No new cases with RAPD type A were observed after implementation of intervention measures that targeted the prevention of transmission via the milking machine as well as improvement of environmental hygiene. A second outbreak of Klebsiella mastitis that occurred several weeks later was caused by multiple RAPD types, which rules out contagious transmission and indicates opportunistic infections originating from the environment. The diversity of Klebsiella strains as quantified with Simpson's index of discrimination was significantly higher for isolates from fecal, feed, and water samples than for isolates from milk samples. Several isolates from bedding material that had the phenotypic appearance of Klebsiella spp. were identified as being Raoultella planticola and Raoultella terrigena based on rpoB sequencing. PMID:17928424

Bacterial dermatosepticemia, a systemic infectious bacterial disease of frogs, can be caused by several opportunistic gram-negative bacterial species including Aeromonas hydrophila, Chryseobacterium indologenes, Chryseobacterium meningosepticum, Citrobacter freundii, Klebsiellapneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia liquifaciens. Here we determined the pathogenicity of 3 bacterial species (Aeromonas hydrophila, Klebsiellapneumoniae, and Proteus mirabilis) associated with an outbreak of fatal dermatosepticemia in New Zealand Litoria ewingii frogs. A bath challenge method was used to expose test frogs to individual bacterial species (2 × 107 cfu/mL in pond water); control frogs were exposed to uninfected pond water. None of the control frogs or those exposed to A. hydrophila or P. mirabilis showed any morbidity or mortality. Morbidity and mortality was 40% among frogs exposed to K. pneumonia, and the organism was reisolated from the hearts, spleens, and livers of affected animals. PMID:20412685

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA can also present in combination with juvenile idiopathic arthritis (JIA), the major chronic rheumatologic disease in children. We report herein the first known case of a juvenile patient diagnosed with XLA combined with JIA that later developed into invasive Klebsiellapneumoniae polyarticular septic polyarthritis. An additional comprehensive review of XLA combined with JIA and invasive K. pneumoniae septic arthritis is also presented. XLA was identified by the detection of BTK mutations while the diagnosis of JIA was established by clinical and laboratory assessments. Septic arthritis caused by invasive K. pneumoniae was confirmed by culturing of the synovia and gene detection of the isolates. Invasive K. pneumoniae infections can not only result in liver abscesses but also septic arthritis, although this is rare. XLA combined with JIA may contribute to invasive K. pneumoniae infection. PMID:24567239

Klebsiellapneumoniae is a colonizer of livestock, a contaminant of retail meats and vegetables, and a cause of extraintestinal infections in humans. Antibiotic-resistant strains of K. pneumoniae are becoming increasingly prevalent among hospital and community-acquired infections. Antibiotics are used extensively in conventional food-animal production, where they select for antibiotic-resistant bacteria. Antibiotic-resistant K. pneumoniae has been isolated from livestock as well as from a variety of retail meats, seafood, and vegetables. Furthermore, recent phylogenetic analyses suggest close relationships between K. pneumoniae from humans and livestock. Therefore, it is essential that we quantify the contribution of foodborne K. pneumoniae to antibiotic-resistant human infections. PMID:27022987

Background: Klebsiellapneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria, which cause serious disease outbreaks worldwide, was rarely detected in Xiangya Hospital, prior to an outbreak that occurred from August 4, 2014, to March 17, 2015. The aim of this study was to analyze the epidemiology and molecular characteristics of the K. pneumoniae strains isolated during the outbreak. Methods: Nonduplicate carbapenem-resistant K. pneumoniae isolates were screened for blaKPC-2 and multiple other resistance determinants using polymerase chain reaction. Subsequent studies included pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, analysis of plasmids, and genetic organization of blaKPC-2 locus. Results: Seventeen blaKPC-2-positive K. pneumoniae were identified. A wide range of resistant determinants was detected. Most isolates (88.2%) coharbored blaKPC-2 and rmtB in addition to other resistance genes, including blaSHV-1, blaTEM-1, and aac(3)-IIa. The blaKPC-2 and rmtB genes were located on the conjugative IncFIB-type plasmid. Genetic organization of blaKPC-2 locusin most strains was consistent with that of the plasmid pKP048. Four types (A1, A2, A3, and B) were detected by PFGE, and Type A1, an ST11, was the predominant PFGE type. A novel K. pneumoniae sequence type (ST1883) related to ST11 was discovered. Conclusions: These isolates in our study appeared to be clonal and ST11 K. pneumoniae was the predominant clone attributed to the outbreak. Coharbing of blaKPC-2 and rmtB, which were located on a transferable plasmid, in clinical K. pneumoniae isolates may lead to the emergence of a new pattern of drug resistance. PMID:27569227

Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and group A Streptoccocus, but no Klebsiellapneumoniae were responsible for bacterial coinfections during the 2009 and previous influenza pandemics. We hereby report a case with concurrent bacteremic pneumonia due to an unusual capsular serotype K16 K. pneumoniae and pandemic (H1N1) 2009 influenza in a patient with nasopharyngeal cancer. Such a coinfection has not previously been described. PMID:22153762

Background: Carbapenem resistance is one of the major threats faced in antimicrobial treatment of infections caused by gram negative organisms. In recent years, carbapenem resistance has emerged in Klebsiellapneumoniae isolates due to acquisition of carbapenemases which belong to Ambler class A KPC type enzymes or to Ambler class B metallo-β-lactamases (MBL). Routine lab detection of carbapenemase producing K. pneumoniae isolates is crucial, both for a therapeutic management and an efficient infection control. Materials and Methods: A study was conducted on 60 carbapenem resistant Klebsiellapneumoniae strains which were isolated from various clinical samples over a period of one year (September 2010-August 2011), at a tertiary care hospital in Jaipur. Phenotypic confirmatory test was done by using discs of Meropenem alone and those with phenyl boronic acid (PBA) or Ethylenediaminetetraacetic acid (EDTA) or both, for detection of carbapenemase production and differentiation of KPC and MBL enzymes. Results: Of the 60 carbapenem resistant Klebsiellapneumoniae isolates, 53 (88.33%) were found to be MBL producers, 4(6.66%) were found to be MBL and KPC co-producers and the rest of the 3(5%) isolates were negative for both MBL and KPC production, as was seen by combined disc testing. Conclusion: The combined disc test is a simple test which can be used for differentiation of carbapenemases and it can be easily incorporated in routine microbiology lab testing. PMID:25177562

Klebsiellapneumoniae has been considered as an important foodborne pathogen which causes severe infections that include meningitis, bronchitis, bacteremia, pneumonia, and urinary tract infections in humans and animals. It is well known to most clinicians as a cause of community-acquired bacterial pneumonia. Klebsiella is an opportunistic pathogen, that primarily attacks neonates, infants, elderly and immuno-compromised patients and therefore impose a serious, emerging public health hazard globally. Contaminated sprouts, vegetables, seafood and other animal meat products are considered as main sources of Klebsiella infection. In the current study, radiation sensitivity of K. pneumoniae MTCC 109 was determined in different food samples. The decimal reduction dose (D10) values of K. pneumoniae MTCC 109 in saline and nutrient broth at 0-4 °C were 0.116±0.009, 0.136±0.005 kGy, respectively. The mixed sprouts, fish and poultry samples were inoculated with K. pneumoniae MTCC 109 and exposed to gamma radiation to evaluate the effectiveness of radiation treatment in the elimination of K. pneumoniae. D10 values of K. pneumoniae in mixed sprouts, poultry and fish samples were found to be 0.142±0.009, 0.125±0.0004 and 0.277±0.012 kGy, respectively. Radiation treatment with a 1.5 kGy dose resulted in the complete elimination of 3.1±1.8×105 CFU/g of K. pneumoniae from these food samples. No recovery of K. pneumoniae was observed in the 1.5 kGy treated samples stored at 4 °C up to 12 days, even after enrichment and selective plating. This study shows that a 1.5 kGy dose of irradiation treatment could lead to the complete elimination of 3.1±1.8×105 CFU/g of K. pneumoniae from mixed sprouts, poultry and fish samples.

Klebsiellapneumoniae is an increasingly important bacterial pathogen to human. This Gram-negative bacterium species has become a serious concern due to its dramatic increase in the levels of multiple antibiotic resistances, particularly to carbapenems. The toxin-antitoxin (TA) system has recently been reported to be involved in the formation of drug-tolerant persister cells. The type II TA system is composed of a stable toxin protein and a relatively unstable antitoxin protein that is able to inhibit the toxin. Here, we examine the type II TA locus distribution and compare the TA diversity throughout ten completely sequenced K. pneumoniae genomes by using bioinformatics approaches. Two hundred and twelve putative type II TA loci were identified in 30 replicons of these K. pneumoniae strains. The amino acid sequence similarity-based grouping shows that these loci distribute differently not only among different K. pneumoniae strains isolated from diverse sources, but also between their chromosomes and plasmids. PMID:26662948

Klebsiellapneumoniae is an opportunistic pathogen that is responsible for causing nosocomial and community-acquired infections. Despite its common presence in soil and aquatic environments, the virulence potential of K. pneumoniae isolates of environmental origin is largely unknown. Hence, in this study, K. pneumoniae isolated from the estuarine waters and sediments of the Matang mangrove estuary were screened for potential virulence characteristics: antibiotic susceptibility, morphotype on Congo red agar, biofilm formation, presence of exopolysaccharide and capsule, possession of virulence genes (fimH, magA, ugE, wabG and rmpA) and their genomic fingerprints. A total of 55 strains of K. pneumoniae were isolated from both human-distributed sites (located along Sangga Besar River) and control sites (located along Selinsing River) where less human activity was observed, indicated that K. pneumoniae is ubiquitous in the environment. However, the detection of potentially virulent strains at the downstream of Kuala Sepetang village has suggested an anthropogenic contamination source. In conclusion, the findings from this study indicate that the Matang mangrove estuary could harbor potentially pathogenic K. pneumoniae with risk to public health. More studies are required to compare the environmental K. pneumoniae strains with the community-acquired K. pneumoniae strains. PMID:27092516

Klebsiellapneumoniae is an opportunistic pathogen that is responsible for causing nosocomial and community-acquired infections. Despite its common presence in soil and aquatic environments, the virulence potential of K. pneumoniae isolates of environmental origin is largely unknown. Hence, in this study, K. pneumoniae isolated from the estuarine waters and sediments of the Matang mangrove estuary were screened for potential virulence characteristics: antibiotic susceptibility, morphotype on Congo red agar, biofilm formation, presence of exopolysaccharide and capsule, possession of virulence genes (fimH, magA, ugE, wabG and rmpA) and their genomic fingerprints. A total of 55 strains of K. pneumoniae were isolated from both human-distributed sites (located along Sangga Besar River) and control sites (located along Selinsing River) where less human activity was observed, indicated that K. pneumoniae is ubiquitous in the environment. However, the detection of potentially virulent strains at the downstream of Kuala Sepetang village has suggested an anthropogenic contamination source. In conclusion, the findings from this study indicate that the Matang mangrove estuary could harbor potentially pathogenic K. pneumoniae with risk to public health. More studies are required to compare the environmental K. pneumoniae strains with the community-acquired K. pneumoniae strains. PMID:27092516

Multidrug-resistant Klebsiellapneumoniae (MRKP) has steadily grown beyond antibiotic control. However, a bacteriophage is considered to be a potential antibiotic alternative for treating bacterial infections. In this study, a lytic bacteriophage, phage 1513, was isolated using a clinical MRKP isolate KP 1513 as the host and was characterized. It produced a clear plaque with a halo and was classified as Siphoviridae. It had a short latent period of 30 min, a burst size of 264 and could inhibit KP 1513 growth in vitro with a dose-dependent pattern. Intranasal administration of a single dose of 2×10(9) PFU/mouse 2 h after KP 1513 inoculation was able to protect mice against lethal pneumonia. In a sublethal pneumonia model, phage-treated mice exhibited a lower level of K. pneumoniae burden in the lungs as compared to the untreated control. These mice lost less body weight and exhibited lower levels of inflammatory cytokines in their lungs. Lung lesion conditions were obviously improved by phage therapy. Therefore, phage 1513 has a great effect in vitro and in vivo, which has potential to be used as an alternative to an antibiotic treatment of pneumonia that is caused by the multidrug-resistant K. pneumoniae. PMID:25879036

Multidrug-resistant Klebsiellapneumoniae (MRKP) has steadily grown beyond antibiotic control. However, a bacteriophage is considered to be a potential antibiotic alternative for treating bacterial infections. In this study, a lytic bacteriophage, phage 1513, was isolated using a clinical MRKP isolate KP 1513 as the host and was characterized. It produced a clear plaque with a halo and was classified as Siphoviridae. It had a short latent period of 30 min, a burst size of 264 and could inhibit KP 1513 growth in vitro with a dose-dependent pattern. Intranasal administration of a single dose of 2 × 109 PFU/mouse 2 h after KP 1513 inoculation was able to protect mice against lethal pneumonia. In a sublethal pneumonia model, phage-treated mice exhibited a lower level of K. pneumoniae burden in the lungs as compared to the untreated control. These mice lost less body weight and exhibited lower levels of inflammatory cytokines in their lungs. Lung lesion conditions were obviously improved by phage therapy. Therefore, phage 1513 has a great effect in vitro and in vivo, which has potential to be used as an alternative to an antibiotic treatment of pneumonia that is caused by the multidrug-resistant K. pneumoniae. PMID:25879036

The use of biologically derived metal nanoparticles for various proposes is going to be an issue of considerable importance; thus, appropriate methods should be developed and tested for the biological synthesis and recovery of these nanoparticles from bacterial cells. In this research study, a strain of Klebsiellapneumoniae was tested for its ability to synthesize elemental selenium nanoparticles from selenium chloride. A broth of Klebsiellapneumoniae culture containing selenium nanoparticles was subjected to sterilization at 121oC and 17 psi for 20 minutes. Released selenium nanoparticles ranged in size from 100 to 550 nm, with an average size of 245 nm. Our study also showed that no chemical changes occurred in selenium nanoparticles during the wet heat sterilization process. Therefore, the wet heat sterilization process can be used successfully to recover elemental selenium from bacterial cells. PMID:24031517

In this study, the discriminatory power of pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods for subtyping of 54 clinical isolates of Klebsiellapneumoniae were compared. All isolates were typeable by RAPD, while 3.6% of them were not typeable by PFGE. The repeatability of both typing methods were 100% with satisfying reproducibility (≥ 95%). Although the discriminatory power of PFGE was greater than RAPD, both methods showed sufficient discriminatory power (DI > 0.95) which reflects the heterogeneity among the K. pneumoniae isolates. An optimized RAPD protocol is less technically demanding and time consuming that makes it a reliable typing method and competitive with PFGE. PMID:24516423

Genome sequencing of carbapenem-resistant Klebsiellapneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the blaKPC genetic context. A phylogeny based on core single-nucleotide variants (SNVs) supports a division of sequence type 258 (ST258) into two distinct groups. The primary differences between the groups are in the capsular polysaccharide locus (cps) and their plasmid contents. A strict association between clade and KPC variant was found. The blaKPC gene was found on variants of two plasmid backbones. This study indicates that highly similar K. pneumoniae subpopulations coexist within the same hospitals over time. PMID:24913165

Broth containing 152 g glycerol l(-1) from Candida krusei culture was converted to 1,3-propanediol by Klebsiellapneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as substrate was developed giving 53 g 1,3-propanediol l(-1) with a yield of 0.41 g g(-1) glycerol and a productivity of 0.94 g l(-1) h(-1). PMID:16912919

The emergence of β-lactamase-producing Enterobacteriaceae in the last few decades has become major challenge faced by hospitals. In this study, isolates of Klebsiellapneumoniae carbapenemase-2 (KPC-2)-producing K. pneumoniae from a tertiary hospital in Mato Grosso do Sul, Brazil, were characterized. Bacterial identification was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF; Bruker Daltonics, Germany) mass spectrometry. The minimum inhibitory concentrations of carbapenems were determined using the agar dilution method as recommended by the Clinical Laboratory Standards Institute guidelines. Carbapenemase production was detected using the modified Hodge test (MHT) and polymerase chain reaction (PCR), followed by DNA sequencing. Of 360 (12.2%) K. pneumoniae isolates obtained between May 2009 and May 2010, 44 (12.2%) were carbapenem nonsusceptible. Of these 44 isolates, thirty-six K. pneumoniae isolates that were positive by MHT and PCR carried the bla KPC-2 gene. Thus, KPC-2producing Klebsiellapneumoniae has been present in a Brazilian hospital located in the Midwest region since at least 2009. PMID:26273265

Klebsiellapneumoniae carbapenemases (KPCs) confer resistance to nearly all β-lactams. This broad-spectrum drug resistance mechanism has rapidly spread in the United States and is reportedly increasing elsewhere in the world. Thus, the emergence of KPC resistance is a major threat to global health. This article reviews the epidemiology and provides an overview of the dissemination of KPC-producing organisms. PMID:23055754

We describe a rare case of Klebsiellapneumoniae renal abscess with metastatic spread leading to endopthalmitis, pulmonary cavitary lesions, and cerebral emboli in a 41-year-old Hispanic female with diabetes mellitus who presented with a four-to-five-day history of fevers, headache, eye pain, and vomiting. She was treated with IV antibiotics and made a gradual but full recovery. PMID:23984128

We report the emergence of a novel VIM variant (VIM-24) in a Klebsiellapneumoniae isolate in Colombia. The isolate displays MICs for carbapenems below the resistance breakpoints, posing a real challenge for its detection. The blaVIM-24 gene was located within a class 1 integron carried on a large plasmid. Further studies are needed to clarify its epidemiological and clinical impact. PMID:21282438

Oxygen sensitivity of the nifLA promoter of Klebsiellapneumoniae has been demonstrated. Studies on the oxygen regulation of nifB-lacZ and nifH-lacZ fusions in the presence of the nifLA operon, which contains either an intact or a deleted nifL gene, indicate that possible both the nifL promoter and the nifL product are responsible for nif repression by oxygen.

In this study we report the analysis of 131 Klebsiellapneumoniae (K. pneumoniae) clinical isolates from patients hospitalized in various wards, of Perugia General Hospital, from August 2014 to January 2015. Forty two isolates (32.1 %), were resistant to at least one carbapenem antibiotic and, among these isolates, 14 (33.3 %) exhibited resistance to colistin. All isolates were carbapenemases producers and 41 (97.6 %) harboured the bla KPC gene. Carbapenem-resistant K. pneumoniae isolates (CRKPs) were, also, typed for the genotypic diversity and the results revealed the circulation of two major clusters.This surveillance study evidences the spread of CRKP isolates in Perugia General Hospital and confirms that carbapenem-resistant K. pneumoniae isolates have reached epidemic dissemination in Italy. In addition the percentage of resistance to colistin resulted to be less than that observed in other hospital laboratories across Italy. In conclusion the circulation of these isolates should be monitored and appropriate policy of surveillance must be used, in a target manner, in order to reduce the spread of carbapenem-resistant isolates. PMID:26810235

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiellapneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters-lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)-were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation. PMID:24858816

Multidrug-resistant (MDR) Klebsiellapneumoniae has become a major contributor to nosocomial bloodstream infections. Here, we report the draft genome sequences of two MDR extended-spectrum-β-lactamase-producing strains causing bloodstream infections. These sequenced genomes display a wide-spectrum virulence arsenal and will help us understand the genomic basis of K. pneumoniae virulence. PMID:26798092

Mediastinitis due to Klebsiellapneumoniae, related to thoracic wall contamination after cardiac surgery, has rarely been described. We aim to report a case of fulminant mediastinitis due to extended-spectrum beta-lactamase-producing K. pneumoniae, secondary to a disseminated concomitant pulmonary infection. The patient remained pauci-symptomatic until clinical manifestations of sepsis acutely appeared. PMID:23416348

We aimed to examine the effects of resistance mechanisms on several resistance phenotypes among carbapenem-resistant Klebsiellapneumoniae isolates with borderline carbapenem MICs. We compared carbapenemase-negative K. pneumoniae with carbapenemase-producing K. pneumoniae (CPKP) isolates with similar MICs. CPKP isolates exhibited a marked inoculum effect and were more resistant to the bactericidal effect of meropenem. This suggests that MIC measurements alone may not be sufficient in predicting the therapeutic efficacy of carbapenems against CPKP. PMID:25987630

We aimed to examine the effects of resistance mechanisms on several resistance phenotypes among carbapenem-resistant Klebsiellapneumoniae isolates with borderline carbapenem MICs. We compared carbapenemase-negative K. pneumoniae with carbapenemase-producing K. pneumoniae (CPKP) isolates with similar MICs. CPKP isolates exhibited a marked inoculum effect and were more resistant to the bactericidal effect of meropenem. This suggests that MIC measurements alone may not be sufficient in predicting the therapeutic efficacy of carbapenems against CPKP. PMID:25987630

Background. Klebsiellapneumoniae is a common colonizer of the gastrointestinal tract of humans, companion animals, and livestock. To better understand potential contributions of foodborne K. pneumoniae to human clinical infections, we compared K. pneumoniae isolates from retail meat products and human clinical specimens to assess their similarity based on antibiotic resistance, genetic relatedness, and virulence. Methods. Klebsiellapneumoniae was isolated from retail meats from Flagstaff grocery stores in 2012 and from urine and blood specimens from Flagstaff Medical Center in 2011–2012. Isolates underwent antibiotic susceptibility testing and whole-genome sequencing. Genetic relatedness of the isolates was assessed using multilocus sequence typing and phylogenetic analyses. Extraintestinal virulence of several closely related meat-source and urine isolates was assessed using a murine sepsis model. Results. Meat-source isolates were significantly more likely to be multidrug resistant and resistant to tetracycline and gentamicin than clinical isolates. Four sequence types occurred among both meat-source and clinical isolates. Phylogenetic analyses confirmed close relationships among meat-source and clinical isolates. Isolates from both sources showed similar virulence in the mouse sepsis model. Conclusions. Meat-source K. pneumoniae isolates were more likely than clinical isolates to be antibiotic resistant, which could reflect selective pressures from antibiotic use in food-animal production. The close genetic relatedness of meat-source and clinical isolates, coupled with similarities in virulence, suggest that the barriers to transmission between these 2 sources are low. Taken together, our results suggest that retail meat is a potential vehicle for transmitting virulent, antibiotic-resistant K. pneumoniae from food animals to humans. PMID:26206847

Klebsiellapneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation. PMID:25776607

Multidrug-resistant (MDR) Klebsiellapneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource-poor hospital settings. Through whole-genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high-dependency wards in a hospital in Kathmandu during 2012 with a case-fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage-specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward-specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM-1 containing lineage in 2011 would have alerted the high-dependency ward staff to intervene. We argue that some form of real-time genetic characterisation, alongside clade-specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high- and low-income settings. PMID:25712531

Invasive Klebsiellapneumoniae with hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that, over the past 20 y, has resulted in a distinct clinical syndrome characterized by pyogenic liver abscesses sometimes complicated by bacteremia, meningitis, and endophthalmitis. Infections occur predominantly in Taiwan and other Asian countries, but HMV K. pneumoniae is considered an emerging infectious disease in the United States and other Western countries. In 2005, fatal multisystemic disease was attributed to HMV K. pneumoniae in African green monkeys (AGM) at our institution. After identification of a cluster of subclinically infected macaques in March and April 2008, screening of all colony nonhuman primates by oropharyngeal and rectal culture revealed 19 subclinically infected rhesus and cynomolgus macaques. PCR testing for 2 genes associated with HMV K. pneumoniae, rmpA and magA, suggested genetic variability in the samples. Random amplified polymorphic DNA analysis on a subset of clinical isolates confirmed a high degree of genetic diversity between the samples. Environmental testing did not reveal evidence of aerosol or droplet transmission of the organism in housing areas. Further research is needed to characterize HMV K. pneumoniae, particularly with regard to genetic differences among bacterial strains and their relationship to human disease and to the apparent susceptibility of AGM to this organism. PMID:20034435

Nosocomial infections, also called “hospital acquired infections,” occur worldwide and affect both developed and resource-poor countries, thus having a major impact on their health care systems. Klebsiellapneumoniae, which is an opportunistic Gram-negative pathogen, is responsible for causing pneumonia, urinary tract infections and septicemia in immune compromised hosts such as neonates. Unfortunately, there is no vaccine or mAb available for prophylactic or therapeutic use against K. pneumoniae infections. For this reason, we sought for a protein-based subunit vaccine capable of combating K. pneumoniae infections, by applying our ANTIGENome technology for the identification of potential vaccine candidates, focusing on conserved protein antigens present in strains with different serotypes. We identified numerous novel immunogenic proteins using genomic surface display libraries and human serum antibodies from donors exposed to or infected by K. pneumoniae. Vaccine candidate antigens were finally selected based on animal protection in a murine lethal-sepsis model. The protective and highly conserved antigens identified in this study are promising candidates for the development of a protein-based vaccine to prevent infection by K. pneumoniae. PMID:23250007

A recombinant cosmid containing genes involved in Klebsiellapneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch β-d-Glc to the O-4 position of l-glycero-d-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica. PMID:11371519

Increasing antibiotic resistance among bacterial pathogens has rendered some infections untreatable with available antibiotics. Klebsiellapneumoniae, a bacterial pathogen that has acquired high-level antibiotic resistance, is a common cause of pulmonary infections. Optimal clearance of K. pneumoniae from the host lung requires TNF and IL-17A. Herein, we demonstrate that inflammatory monocytes are rapidly recruited to the lungs of K. pneumoniae-infected mice and produce TNF, which markedly increases the frequency of IL-17-producing innate lymphoid cells. While pulmonary clearance of K. pneumoniae is preserved in neutrophil-depleted mice, monocyte depletion or TNF deficiency impairs IL-17A-dependent resolution of pneumonia. Monocyte-mediated bacterial uptake and killing is enhanced by ILC production of IL-17A, indicating that innate lymphocytes engage in a positive-feedback loop with monocytes that promotes clearance of pneumonia. Innate immune defense against a highly antibiotic-resistant bacterial pathogen depends on crosstalk between inflammatory monocytes and innate lymphocytes that is mediated by TNF and IL-17A. PMID:27040495

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiellapneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2. PMID:1696932

Objective To investigate the genotype of klebsiellapneumonia strains isolated from eldly inpatients by multiple-locus variable-number tandem-repeat analysis. Methods Totally 184 klebsiellapneumonia strains,isolated from eldly inpatients,were collected,and their genome DNA were extracted. The polymorphism of 7 variable-number tandem-repeat locus in the DNA samples was analyzed by multiple primers polymerase chain reaction and capillary electrophoresis. The clustering analysis of genotyping was carried out with the BioNumerics 5.1 software. Results A total of 139 genotypes were identified in 184 klebsiellapneumonia clinical strains,showing obvious genetic polymorphisms. With clustering analysis of genotypes,all the strains were categorized into three gene clusters (genogroups 1,2,and 3). The genogroup 1 was the biggest cluster,containing 93.06% of the isolated strains. Conclusion There was a predominant cluster in the klebsiellapneumonia strains isolated from eldly inpatients in our center,and the major source of klebsiellapneumonia infection remained the nosocomial infection. PMID:27594157

The aim of the present study was to investigate the phenotype and genotype of plasmid-mediated AmpC (pAmpC) β-lactamase in Klebsiellapneumoniae and its antibiotic resistance. A total of 130 non-repetitive clinical isolates of Klebsiellapneumoniae, obtained from tertiary hospitals, were phenotypically screened for pAmpC β-lactamase production with the cefoxitin disk diffusion test. β-lactamase genes in the screened isolates were detected using multiplex polymerase chain reaction (PCR); carbapenemase genes in pAmpC β-lactamase-producing isolates that were resistant to imipenem were detected using PCR. Out of the 130 isolates of Klebsiellapneumoniae, 62 strains (47.7%) were resistant to cefoxitin, including 14 strains (10.8%) positive for pAmpC β-lactamase (DHA type), among which 12 strains (85.7%) were susceptible to imipenem, and 2 strains, which were carrying Klebsiellapneumoniae carbapenemase (KPC)-2 gene, were resistant to imipenem. The pAmpC β-lactamase-producing Klebsiellapneumoniae isolates from the tertiary hospitals were mainly of DHA-1 genotype, and the majority were susceptible to carbapenems; drug-resistant strains were associated with KPC-2 expression. PMID:27347082

Background Klebsiellapneumoniae-caused liver abscess (KLA) has become a health problem in Taiwan and is continually reported in other countries. Diabetes mellitus, the most common metabolic disorder, underlies half of the KLA patients in Taiwan. The clinical impact of KLA has been well-documented. Nevertheless, the molecular basis regarding how K. pneumoniae causes liver infection, particularly in diabetic individuals, remains unclear. Methodology/Principle Findings Auto-bioluminescence-expressing K. pneumoniae was inoculated into diabetic mice and age-match naïve control. With the use of in vivo imaging system, translocation of the bioluminescence-expressing K. pneumoniae from intestine to extraintestinal organs, mainly the liver, was noted in 80% of the diabetic mice, whereas the same bacteria causes extraintestinal infections in only 31% of naïve mice. Besides increased morbidity, the severity of hepatic tissue injury was also enhanced in the K. pneumoniae-infected diabetic mice. Upon K. pneumoniae infection, IFN-γ production was significantly evoked in the liver. To mediate IFN-γ signal, STAT (signal transducers and activators of transcription) 1 and 3 were activated in hepatocytes, and so was the expression of IRF (interferon regulatory factor)-1. Moreover, accumulation of neutrophils which was triggered by prolonged production of IL-1β and MIP-2, and significant increases in the level of active caspase 3 and phospho-eIF2α, were exclusively revealed in the K. pneumoniae-infected diabetic mice. Conclusion The activation of IFN-γ/STAT/IRF-1 signaling demonstrated by this work emphasizes the role of IFN-γ for mediating the hepatic response to K. pneumoniae infection. PMID:24223208

This study describes the molecular characteristics and risk factors associated with carbapenem-resistant Klebsiellapneumoniae strains. Risk factors associated with KPC-producing K. pneumoniae strains were investigated in this case-control study from May 2011 to May 2013. Bacterial identification was performed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined by broth microdilution. Carbapenemase production was assessed by both modified Hodge test (MHT) and ertapenem hydrolysis using MALDI-TOF MS. The presence of β-lactamase-encoding genes was evaluated by PCR and DNA sequencing. Alterations in genes encoding K. pneumoniae outer membrane proteins were analysed by PCR and DNA sequencing as well as SDS-PAGE. Genetic relatedness among strains was determined by pulsed-field gel electrophoresis. This study included 94 patients. Longer hospitalisation, mechanical ventilation, catheters, and previous surgery were associated with KPC-producing K. pneumoniae. Sixty-eight strains showed resistance to carbapenems. Carbapenemase production was detected by MHT in 67 K. pneumoniae strains and by MALDI-TOF MS in 57. The presence of the blaKPC-2 gene was identified in 57 strains. The blaKPC-2 gene was not found in 11 carbapenem-resistant K. pneumoniae; instead, the blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8 like, blaCTX-M-14-like and blaSHV- like genes associated with OmpK35 and OmpK36 alterations were observed. Thirty-three KPC-producing K. pneumoniae strains were clonally related, and patients infected with these strains had a higher mortality rate (78.78 %). Our results show that KPC-producing K. pneumoniae was associated with several healthcare-related risk factors, including recent surgery. PMID:27002853

The emergence of carbapenem-resistant Gram-negative pathogens poses a serious threat to public health worldwide. In particular, the increasing prevalence of carbapenem-resistant Klebsiellapneumoniae is a major source of concern. K. pneumoniae carbapenemases (KPCs) and carbapenemases of the oxacillinase-48 (OXA-48) type have been reported worldwide. New Delhi metallo-β-lactamase (NDM) carbapenemases were originally identified in Sweden in 2008 and have spread worldwide rapidly. In this review, we summarize the epidemiology of K. pneumoniae producing three carbapenemases (KPCs, NDMs, and OXA-48-like). Although the prevalence of each resistant strain varies geographically, K. pneumoniae producing KPCs, NDMs, and OXA-48-like carbapenemases have become rapidly disseminated. In addition, we used recently published molecular and genetic studies to analyze the mechanisms by which these three carbapenemases, and major K. pneumoniae clones, such as ST258 and ST11, have become globally prevalent. Because carbapenemase-producing K. pneumoniae are often resistant to most β-lactam antibiotics and many other non-β-lactam molecules, the therapeutic options available to treat infection with these strains are limited to colistin, polymyxin B, fosfomycin, tigecycline, and selected aminoglycosides. Although, combination therapy has been recommended for the treatment of severe carbapenemase-producing K. pneumoniae infections, the clinical evidence for this strategy is currently limited, and more accurate randomized controlled trials will be required to establish the most effective treatment regimen. Moreover, because rapid and accurate identification of the carbapenemase type found in K. pneumoniae may be difficult to achieve through phenotypic antibiotic susceptibility tests, novel molecular detection techniques are currently being developed. PMID:27379038

The emergence of carbapenem-resistant Gram-negative pathogens poses a serious threat to public health worldwide. In particular, the increasing prevalence of carbapenem-resistant Klebsiellapneumoniae is a major source of concern. K. pneumoniae carbapenemases (KPCs) and carbapenemases of the oxacillinase-48 (OXA-48) type have been reported worldwide. New Delhi metallo-β-lactamase (NDM) carbapenemases were originally identified in Sweden in 2008 and have spread worldwide rapidly. In this review, we summarize the epidemiology of K. pneumoniae producing three carbapenemases (KPCs, NDMs, and OXA-48-like). Although the prevalence of each resistant strain varies geographically, K. pneumoniae producing KPCs, NDMs, and OXA-48-like carbapenemases have become rapidly disseminated. In addition, we used recently published molecular and genetic studies to analyze the mechanisms by which these three carbapenemases, and major K. pneumoniae clones, such as ST258 and ST11, have become globally prevalent. Because carbapenemase-producing K. pneumoniae are often resistant to most β-lactam antibiotics and many other non-β-lactam molecules, the therapeutic options available to treat infection with these strains are limited to colistin, polymyxin B, fosfomycin, tigecycline, and selected aminoglycosides. Although, combination therapy has been recommended for the treatment of severe carbapenemase-producing K. pneumoniae infections, the clinical evidence for this strategy is currently limited, and more accurate randomized controlled trials will be required to establish the most effective treatment regimen. Moreover, because rapid and accurate identification of the carbapenemase type found in K. pneumoniae may be difficult to achieve through phenotypic antibiotic susceptibility tests, novel molecular detection techniques are currently being developed. PMID:27379038

Background and Objectives: The rapid emergence and dissemination of carbapenemase-producing Klebsiellapneumoniae strains and other members of the Enterobacteriaceae poses a considerable threat to the care of hospitalized patients and to public health. The aim of this study was to determine the frequency of metallo-β-lactamases (MBL) and VIM-1 gene in multidrug-resistant strains of K. pneumoniae. Methods: 50 isolates of non – duplicated K. pneumoniae cultured from patients at intensive care units were tested for their susceptibilities to 13 different antibiotics using microbroth dilution assay. Isolates showing resistance to at least one of the carbapenems were checked for production of metallo-β-lactamase (MBLs) using imipenem–EDTA synergy tests. PCR was used to detect the gene encoding VIM-1 metallo-β-lactamase (MBL). Results: Of 50 clinical isolates, 26 (52%) were resistant to imipenem in disk diffusion method. Using imipenem–EDTA synergy tests, production of MBL was detected in 15 (30%) isolates. PCR assay showed that 15 isolates were positive for VIM and these included 10 and 5 isolates showing positive and negative results in phenotypic method of MBL detection test respectively. Amikacin was found as the most effective antibiotic against the MBL producers in this study. Conclusion: The emergence of bla(VIM-1) producing K. pneumoniae in North of Iran is concerning. Microorganisms producing bla(VIM-1) constitute the prevalent multidrug-resistant population of K. pneumoniae in that region. PMID:26622969

Klebsiellapneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins. PMID:25633080

Klebsiellapneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins. PMID:25633080

Multidrug-resistant and highly virulent Klebsiellapneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected. PMID:25341126

OBJECTIVE: Enterobacteriaceae bacteria harboring Klebsiellapneumoniae carbapenemase are a serious worldwide threat. The molecular identification of these pathogens is not routine in Brazilian hospitals, and a rapid phenotypic screening test is desirable. This study aims to evaluate the modified Hodge test as a phenotypic screening test for Klebsiellapneumoniae carbapenemase. METHOD: From April 2009 to July 2011, all Enterobacteriaceae bacteria that were not susceptible to ertapenem according to Vitek2 analysis were analyzed with the modified Hodge test. All positive isolates and a random subset of negative isolates were also assayed for the presence of blaKPC. Isolates that were positive in modified Hodge tests were sub-classified as true-positives (E. coli touched the ertapenem disk) or inconclusive (distortion of the inhibition zone of E. coli, but growth did not reach the ertapenem disk). Negative results were defined as samples with no distortion of the inhibition zone around the ertapenem disk. RESULTS: Among the 1521 isolates of Enterobacteriaceae bacteria that were not susceptible to ertapenem, 30% were positive for blaKPC, and 35% were positive according to the modified Hodge test (81% specificity). Under the proposed sub-classification, true positives showed a 98% agreement with the blaKPC results. The negative predictive value of the modified Hodge test for detection was 100%. KPC producers showed high antimicrobial resistance rates, but 90% and 77% of these isolates were susceptible to aminoglycoside and tigecycline, respectively. CONCLUSION: Standardizing the modified Hodge test interpretation may improve the specificity of KPC detection. In this study, negative test results ruled out 100% of the isolates harboring Klebsiellapneumoniae carbapenemase-2. The test may therefore be regarded as a good epidemiological tool. PMID:23295597

Emergence and spread of carbapenemases in Enterobacteriaceae is a cause of concern worldwide, the latest threat being New Delhi metallo-β-lactamase (NDM-1). This report is of an orthopedic case with fracture femur managed with internal fixation and bone grafting, who subsequently developed secondary infection with Klebsiellapneumoniae harboring blaNDM-1 gene. Minimum inhibitory concentration (MIC) of imipenem was ≥8 μg/ml by E-test, suggestive of carbapenemase production. Phenotypic and further genotypic detection confirmed the presence of blaNDM-1 gene. The isolate remained susceptible only to tigecycline, colistin, and polymyxin B. PMID:25298566

We describe a clinical case of an abdominal abscess due to NDM-1-producing Klebsiellapneumoniae in a 35-year-old Spanish patient after hospitalization in India for perforated appendicitis and peritonitis. The strain belonged to the MLST type 231 and had multiple additional antibiotic resistance genes such as bla(CTX-M-15), armA methylase, aac(6')-Ib-cr, dfrA12, sul1 and qnrB and lack of porin genes ompK35 and ompK36. The patient was cured after abscess drainage. PMID:22383442

We report the emergence of five carbapenem-resistant K1 hypervirulent Klebsiellapneumoniae (hvKP) strains which caused fatal infections in hospital patients in Zhejiang Province, China, upon entry through surgical wounds. Genotyping results revealed the existence of three genetically related strains which exhibited a new sequence type, ST1797, and revealed that all strains harbored the magA and wcaG virulence genes and a plasmid-borne blaKPC-2 gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible. PMID:26574010

Both the physical behavior of aerosols and survival of airborne Serratia marcescens in hyperbaric chambers with a helium-air mixture at 20 atm of pressure was approximately the same as in the system at ambient pressures. Exposure of mice to aerosols of Klebsiellapneumoniae at 1-, 2-, and 17-atm (ca. 101-, 203-, and 1,722-kPa) pressures of helium-oxygen mixture showed that the number of viable organisms constituting a 50% lethal dose was not significantly affected by the hyperbaric conditions. Images PMID:6996616

Septic arthritis is encountered very rarely during the neonatal period and its diagnosis can delay because of atypical symptoms, thus it may lead to serious sequelae. The sequale can be prevented by early diagnosis and concomitant treatment. In neonates, pain can be experienced as a result of pseudoparalysis and of movement of the effected joints. A 17-day-old neonatal patient was brought to our hospital with complaint of unrest and then diagnosed with septic arthritis due to propagation of Klebsiellapneumoniae in joint fluid culture was represented because of the rarity of such a case. PMID:27042550

Many plants, fungi, and bacteria catabolize allantoin as a mechanism for nitrogen assimilation. Recent reports have shown that in plants and some bacteria the product of hydrolysis of allantoin by allantoinase is the unstable intermediate ureidoglycine. While this molecule can spontaneously decay, genetic analysis of some bacterial genomes indicates that an aminotransferase may be present in the pathway. Here we present evidence that Klebsiellapneumoniae HpxJ is an aminotransferase that preferentially converts ureidoglycine and an {alpha}-keto acid into oxalurate and the corresponding amino acid. We determined the crystal structure of HpxJ, allowing us to present an explanation for substrate specificity.

Six multiresistant, NDM-1-producing Klebsiellapneumoniae strains were recovered from an outbreak that affected six neonatal patients in a Colombian hospital. Molecular analysis showed that all of the isolates harbored the blaNDM-1, qnrA, and intI1 genes and were clonally related. Multilocus sequence typing showed that the isolates belonged to a new sequence type (ST1043) that was different from the sequence types that had previously been reported. This is the first report of NDM-1-producing isolates in South America. PMID:23357776

Two genetically unrelated OXA-163-carrying Klebsiellapneumoniae strains were identified from two infection cases in June 2009 and May 2010 in Cairo, Egypt. OXA-163-producing Enterobacteriaceae had been previously reported in Argentina only. Both patients had no history of travel abroad. The emergence of this newly recognized OXA-48-related β-lactamase able to hydrolyze cephalosporins and carbapenems is especially worrying in a geographic area where OXA-48 is endemic and effective surveillance for antibiotic resistance is largely unaffordable. PMID:22518851

This case report describes a 37-year-old woman who develops an intraparenchymal hepatic haematoma after an endoscopic retrograde cholangiopancreatography with papillotomy and stone extraction. The procedure requires the passage of a guidewire. The patient develops acute abdominal pain 72 hours later and a magnetic resonance shows a hematoma of 124 x 93 mm. She remains under observation. Twenty one days later she complains of upper right abdominal pain and fever. Consequently, a percutaneous drainage is performed isolating Citrobacter freundii and Klebsiellapneumoniae BLEE. The patient has a good evolution. PMID:25199307

In Asia, bla(KPC) detection has been limited to East Asia and not yet seen in Southeast Asia. We report four bla(KPC-2)-containing Klebsiellapneumoniae isolates from two different hospitals in Singapore. All isolates belonged to strain type 11 (ST11) and were indistinguishable by pulsed-field gel electrophoresis (PFGE). bla(KPC-2) was located on nonconjugative plasmids and flanked by mobile genetic structures composed of a partial Tn4401 transposon and a Tn3-based transposon which previously have been described only in Chinese isolates. PMID:22116160

Performance of Vitek2, Etest, and TREK broth microdilution (BMD) panels was evaluated versus reference CLSI BMD for gentamicin susceptibility testing with 57 bloodstream isolates of KPC-producing Klebsiellapneumoniae. Compared with reference BMD, the Essential Agreement and Categorical Agreement for TREK panels, Vitek2, and Etest were 91.2%, 31.6%, and 61.4%, respectively, and 86%, 21%, and 52.6%, respectively. Four very major discrepancies occurred with Vitek2. In these 4 strains, gentamicin resistance was associated with the presence of an armA aminoglycoside resistance determinant. PMID:25533616

An organic acid extracted from purified dinitrogenase isolated from a nifV mutant of Klebsiellapneumoniae has been identified as citric acid. H{sub 2} evolution by the citrate-containing dinitrogenase is partially inhibited by CO, and by some substrates for nitrogenase. The response of maximum velocities to changes in pH for both the wild-type and the NifV{sup {minus}} dinitrogenase was compared. No substantial differences between the enzymes were observed, but there are minor differences. Both enzymes are stable in the pH range 4.8-10, but the enzyme activities dropped dramatically below pH 6.2.

Intragastric Klebsiellapneumoniae infections of mice can cause liver abscesses, necrosis of liver tissues, and bacteremia. Lithium chloride, a widely prescribed drug for bipolar mood disorder, has been reported to possess anti-inflammatory properties. Using an intragastric infection model, the effects of LiCl on K. pneumoniae infections were examined. Providing mice with drinking water containing LiCl immediately after infection protected them from K. pneumoniae-induced death and liver injuries, such as necrosis of liver tissues, as well as increasing blood levels of aspartate aminotransferase and alanine aminotransferase, in a dose-dependent manner. LiCl administered as late as 24 h postinfection still provided protection. Monitoring of the LiCl concentrations in the sera of K. pneumoniae-infected mice showed that approximately 0.33 mM LiCl was the most effective dose for protecting mice against infections, which is lower than the clinically toxic dose of LiCl. Surveys of bacterial counts and cytokine expression levels in LiCl-treated mice revealed that both were effectively inhibited in blood and liver tissues. Using in vitro assays, we found that LiCl (5 μM to 1 mM) did not directly interfere with the growth of K. pneumoniae but made K. pneumoniae cells lose the mucoid phenotype and become more susceptible to macrophage killing. Furthermore, low doses of LiCl also partially enhanced the bactericidal activity of macrophages. Taken together, these data suggest that LiCl is an alternative therapeutic agent for K. pneumoniae-induced liver infections. PMID:25534739

Objectives Klebsiellapneumoniae is a gram-negative rod bacterium, a known cause of community-acquired bacterial pneumonia and is an important hospital-acquired pathogen that causes severe morbidity and mortality. The aim of this study was to identify the TEM and SHV genes in K. pneumoniae isolated from cockroaches obtained from hospitals. Methods In this study, 250 cockroaches were collected from different hospitals in the province of Chaharmahal Va Bakhtiari, which is located in southwest Iran. The samples were examined for the presence of K. pneumoniae by plating onto a combination of culture media, and the antimicrobial susceptibility patterns of isolated K. pneumoniae from samples were evaluated using the disk diffusion test. In addition, from the culture, genomic bacterial DNA was extracted, and sequence-specific targets (TEM and SHV genes) were amplified using the polymerase chain reaction (PCR) method. Results Out of 250 cockroach samples collected from various hospitals, 179 samples (71.60%) were positive for K. pneumoniae. PCR reaction was performed using specific oligonucleotide primers (TEM-F, TEM-R and SHV-F, SHV-R) for the amplification of each gene, and amplified products were visualized on 1% agarose gel electrophoresis. Of all the specimens amplified by PCR in this research, 32 samples (17.87%) were positive for TEM and 15 samples (8.37%) were positive for SHV. Conclusion Detection of TEM and SHV genes using molecular methods and their pattern of antimicrobial resistance can provide useful information about the epidemiology of and risk factors associated with K. pneumoniae infection. PMID:25737824

The objective of this study was to determine whether acquisition of New Delhi metallo-β-lactamase-1 (NDM-1) has an impact on the fitness and virulence of Escherichia coli and Klebsiellapneumoniae. Growth kinetics and the cost of fitness of NDM-1 plasmid carriage were assessed in isogenic E. coli J53 and K. pneumoniae PRZ in vitro by pairwise competition assays. The pathogenicity of NDM-1-expressing E. coli and K. pneumoniae strains and their isogenic controls was analysed in vivo using a Galleria mellonella infection model. The cytotoxicity of NDM-1 was assessed in A549 human lung epithelial cells using the lactate dehydrogenase (LDH) assay. No differences in growth kinetics were recorded between NDM-1-expressing strains and controls (P = 0.92). A reduction in fitness of NDM-1-carrying strains was observed both for E. coli J53 and K. pneumoniae PRZ [selection rate constant (s) = -1.27 ± 0.27 for E. coli J53 and -0.19 ± 0.14 for K. pneumoniae PRZ; P pneumoniae PRZ. Cytotoxicity in A549 cells was not affected by NDM-1 expression (P > 0.05). The presence of blaNDM-1 did not increase the virulence or cytotoxicity of isogenic strains. However, there was a considerable cost of fitness incurred by carriage of the pNDM-1 plasmid. Interestingly, the cost of fitness was significantly higher in E. coli J53 compared with K. pneumoniae PRZ. PMID:27179815

Patients infected or colonized with carbapenem-resistant Klebsiellapneumoniae (CRKp) are often chronically and acutely ill, which results in substantial mortality unrelated to infection. Therefore, estimating excess mortality due to CRKp infections is challenging. The Consortium on Resistance against Carbapenems in K. pneumoniae (CRACKLE) is a prospective multicenter study. Here, patients in CRACKLE were evaluated at the time of their first CRKp bloodstream infection (BSI), pneumonia or urinary tract infection (UTI). A control cohort of patients with CRKp urinary colonization without CRKp infection was constructed. Excess hospital mortality was defined as mortality in cases after subtracting mortality in controls. In addition, the adjusted hazard ratios (aHR) for time-to-hospital-mortality at 30 days associated with infection compared with colonization were calculated in Cox proportional hazard models. In the study period, 260 patients with CRKp infections were included in the BSI (90 patients), pneumonia (49 patients) and UTI (121 patients) groups, who were compared with 223 controls. All-cause hospital mortality in controls was 12%. Excess hospital mortality was 27% in both patients with BSI and those with pneumonia. Excess hospital mortality was not observed in patients with UTI. In multivariable analyses, BSI and pneumonia compared with controls were associated with aHR of 2.59 (95% CI 1.52-4.50, p <0.001) and 3.44 (95% CI 1.80-6.48, p <0.001), respectively. In conclusion, in patients with CRKp infection, pneumonia is associated with the highest excess hospital mortality. Patients with BSI have slightly lower excess hospital mortality rates, whereas excess hospital mortality was not observed in hospitalized patients with UTI. PMID:26850824

Intragastric Klebsiellapneumoniae infections of mice can cause liver abscesses, necrosis of liver tissues, and bacteremia. Lithium chloride, a widely prescribed drug for bipolar mood disorder, has been reported to possess anti-inflammatory properties. Using an intragastric infection model, the effects of LiCl on K. pneumoniae infections were examined. Providing mice with drinking water containing LiCl immediately after infection protected them from K. pneumoniae-induced death and liver injuries, such as necrosis of liver tissues, as well as increasing blood levels of aspartate aminotransferase and alanine aminotransferase, in a dose-dependent manner. LiCl administered as late as 24 h postinfection still provided protection. Monitoring of the LiCl concentrations in the sera of K. pneumoniae-infected mice showed that approximately 0.33 mM LiCl was the most effective dose for protecting mice against infections, which is lower than the clinically toxic dose of LiCl. Surveys of bacterial counts and cytokine expression levels in LiCl-treated mice revealed that both were effectively inhibited in blood and liver tissues. Using in vitro assays, we found that LiCl (5 μM to 1 mM) did not directly interfere with the growth of K. pneumoniae but made K. pneumoniae cells lose the mucoid phenotype and become more susceptible to macrophage killing. Furthermore, low doses of LiCl also partially enhanced the bactericidal activity of macrophages. Taken together, these data suggest that LiCl is an alternative therapeutic agent for K. pneumoniae-induced liver infections. PMID:25534739

Klebsiellapneumoniae (K. pneumoniae) is one of the major pathogen associated with nosocomial infections, especially catheter associated urinary tract infections which involved biofilm formation. This study was designed to evaluate the antibiofilm efficacy of gold nanoparticle conjugated with chlorhexidine (Au-CHX) against K. pneumoniae isolates. Au-CHX was synthesized and analyzed for stability by using UV-Visible spectrophotometry, atomic force microscopy (AFM), fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectroscopy (ESI-MS). Biofilm inhibition and eradication was performed by crystal violet, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and further confirmed by florescence and AFM microscopy. Au-CHX showed the maxima surface plasmon resonance (SPR) band at 535 nm, spherical morphology and polydispersity with size in the range of 20-100 nm. The micro molar concentrations (i.e. 25 and 100 μM) of Au-CHX completely inhibited the biofilm formation and metabolic activity within biofilms of K. pneumoniae reference and three tested clinical isolates, respectively. Time dependant biofilm inhibition assay showed that Au-CHX inhibited the early stage of biofilm formation. While at 75 and 100 μM concentrations, it also eradicated the established biofilms of K. pneumoniae isolates as compared to 2 mM chlorhexidine. Reduced florescence signals and surface roughness during microscopic analysis further confirms the antibiofilm activity of Au-CHX against K. pneumoniae ATCC13882 and clinical isolates. Thus it is concluded that chlorhexidine coated gold nanoparticle not only inhibits the biofilm formation of K. pneumoniae ATCC and clinical isolates but also eradicated the preformed biofilm. PMID:27321770

The aim of the present study was to investigate the presence of extended-spectrum beta-lactamases (ESBL) in Klebsiellapneumoniae isolated at the "Hospital de Niños de Córdoba". The strains were collected from inpatients between January 1996 and July 2000. A total of 150 ESBL producer isolates were detected. During 1996 the prevalence of ESBL producer K. pneumoniae was 20%, but since 1998 the values have increased to approximately 60%. Phenotypic analysis such as isoelectric point (pl) and antibiotyping performed in 32 randomly selected isolates showed two different enzyme profiles: 81% had ESBL with pl = 7.9 and preferential activity against cefotaxime, while 19% showed ESBL with pl = 5.4 and preferential activity against ceftazidime. No isolates resistant to imipenem or ciprofloxacin were detected. Susceptibility to other antimicrobial agents varied, but resistance to gentamicin was strongly associated with ESBL producer isolates. Resistance determinants could be transferred to Escherichia coli by conjugation assays. PMID:12833674

2-Butanone is an important commodity chemical of wide application in different areas. In this study, Klebsiellapneumoniae was engineered to directly produce 2-butanone from glucose by extending its native 2, 3-butanediol synthesis pathway. To identify the potential enzyme for the efficient conversion of 2, 3-butanediol to 2-butanone, we screened different glycerol dehydratases and diol dehydratases. By introducing the diol dehydratase from Lactobacillus brevis and deleting the ldhA gene encoding lactate dehydrogenase, the engineered K. pneumoniae was able to accumulate 246 mg/L of 2-butanone in shake flask. With further optimization of culture condition, the titer of 2-butanone was increased to 450 mg/L. This study lays the basis for developing an efficient biological process for 2-butanone production. PMID:26465746

The ability of Klebsiellapneumoniae strains to resist the bactericidal activity of serum was quantitated. The K. pneumoniae strains tested included mutants lacking the capsular polysaccharide and mutants having a modified lipopolysaccharide structure. The last mutants were obtained as phage-resistant mutants, and their lipopolysaccharide was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chemical analysis. Serum-resistant mutants derived from phage-resistant mutants (lipopolysaccharide mutants) were also characterized. Resistance to the bactericidal activity of complement was mediated by the lipopolysaccharide, especially by the O-antigen polysaccharide chains. The capsular polysaccharide seemed not to play any important role in resistance to serum bactericidal activity in this bacterium. Images PMID:3531020

The aim of this work was to compare the effects of electrosynthesis on different bacterial species. The effects of neutral red-mediated electrosynthesis on the metabolite profiles of three microorganisms: Escherichia coli, Klebsiellapneumoniae, and Zymomonas mobilis, were measured and compared and contrasted. A statistically comprehensive analysis of neutral red-mediated electrosynthesis is presented using the analysis of end-product profiles, current delivered, and changes in cellular protein expression. K. pneumoniae displayed the most dramatic response to electrosynthesis of the three bacteria, producing 93% more ethanol and 76% more lactate vs. control fermentation with no neutral red and no electron delivery. Z. mobilis showed no response to electrosynthesis except elevated acetate titers. Stoichiometric comparison showed that NAD(+) reduction by neutral red could not account for changes in metabolites during electrosynthesis. Neutral red-mediated electrosynthesis was shown to have multifarious effects on the three species. PMID:26096579

Lemierre's syndrome is characterized by an oropharyngeal infection with internal jugular vein thrombosis followed by metastatic infections in other organs. This infection is usually caused by Fusobacterium spp. In this report, we present a rare case of Klebsiellapneumoniae-associated Lemierre's syndrome in a patient with poorly-controlled diabetes mellitus. The infection was complicated by septic emboli in many organs, which led to the patient's death, despite combined antibiotics, anticoagulant therapy, and surgical intervention. Therein, a literature review was performed for reported cases of Lemierre's syndrome caused by Klebsiellapneumoniae and the results are summarized here. PMID:26279962

Lemierre's syndrome is characterized by an oropharyngeal infection with internal jugular vein thrombosis followed by metastatic infections in other organs. This infection is usually caused by Fusobacterium spp. In this report, we present a rare case of Klebsiellapneumoniae-associated Lemierre's syndrome in a patient with poorly-controlled diabetes mellitus. The infection was complicated by septic emboli in many organs, which led to the patient's death, despite combined antibiotics, anticoagulant therapy, and surgical intervention. Therein, a literature review was performed for reported cases of Lemierre's syndrome caused by Klebsiellapneumoniae and the results are summarized here. PMID:26279962

Klebsiellapneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved. PMID:27050128

Klebsiellapneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved. PMID:27050128

Klebsiellapneumonia usually causes urinary tract infections, pneumonia, and other infectious diseases in hospitalized and immunocompromised patients. Among the types of Klebsiellapneumonia, serotype K1 is known to be a highly virulent pathogen. We herein report the case of a healthy 63-year-old man with a pyogenic liver abscess and bilateral endogenous endophthalmitis caused by serotype K1 Klebsiellapneumonia. Although the patient received percutaneous abscess drainage and antibiotic therapy, he lost his eyesight. To improve the poor prognoses of ocular complications, providing both an earlier diagnosis and treatment is critical. PMID:23583997

Klebsiellapneumoniae is responsible for a wide range of clinical symptoms. How this bacterium adapts itself to ever-changing host milieu is still a mystery. Recently, small non-coding RNAs (sRNAs) have received considerable attention for their functions in fine-tuning gene expression at a post-transcriptional level to promote bacterial adaptation. Here we demonstrate that Hfq, an RNA-binding protein, which facilitates interactions between sRNAs and their mRNA targets, is critical for K. pneumoniae virulence. A K. pneumoniae mutant lacking hfq (Δhfq) failed to disseminate into extra-intestinal organs and was attenuated on induction of a systemic infection in a mouse model. The absence of Hfq was associated with alteration in composition of envelope proteins, increased production of capsular polysaccharides, and decreased resistance to H2O2, heat shock, and UV irradiation. Microarray-based transcriptome analyses revealed that 897 genes involved in numerous cellular processes were deregulated in the Δhfq strain. Interestingly, Hfq appeared to govern expression of many genes indirectly by affecting sigma factor RpoS and RpoE, since 19.5% (175/897) and 17.3% (155/897) of Hfq-dependent genes belong to the RpoE- and RpoS-regulon, respectively. These results indicate that Hfq regulates global gene expression at multiple levels to modulate the physiological fitness and virulence potential of K. pneumoniae. PMID:21779404

Klebsiellapneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055ΔlpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055ΔlpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins. PMID:17371870

3-Hydroxypropionic acid (3-HP) is an important platform chemical proposed by the United States Department of Energy. 3-HP can be converted to a series of bulk chemicals. Biological production of 3-HP has made great progress in recent years. However, low yield of 3-HP restricts its commercialization. In this study, systematic optimization was conducted towards high-yield production of 3-HP in Klebsiellapneumoniae. We first investigated appropriate promoters for the key enzyme (aldehyde dehydrogenase, ALDH) in 3-HP biosynthesis, and found that IPTG-inducible tac promoter enabled overexpression of an endogenous ALDH (PuuC) in K. pneumoniae. We optimized the metabolic flux and found that blocking the synthesis of lactic acid and acetic acid significantly increased the production of 3-HP. Additionally, fermentation conditions were optimized and scaled-up cultivation were investigated. The highest 3-HP titer was observed at 83.8 g/L with a high conversion ratio of 54% on substrate glycerol. Furthermore, a flux distribution model of glycerol metabolism in K. pneumoniae was proposed based on in silico analysis. To our knowledge, this is the highest 3-HP production in K. pneumoniae. This work has significantly advanced biological production of 3-HP from renewable carbon sources. PMID:27230116

3-Hydroxypropionic acid (3-HP) is an important platform chemical proposed by the United States Department of Energy. 3-HP can be converted to a series of bulk chemicals. Biological production of 3-HP has made great progress in recent years. However, low yield of 3-HP restricts its commercialization. In this study, systematic optimization was conducted towards high-yield production of 3-HP in Klebsiellapneumoniae. We first investigated appropriate promoters for the key enzyme (aldehyde dehydrogenase, ALDH) in 3-HP biosynthesis, and found that IPTG-inducible tac promoter enabled overexpression of an endogenous ALDH (PuuC) in K. pneumoniae. We optimized the metabolic flux and found that blocking the synthesis of lactic acid and acetic acid significantly increased the production of 3-HP. Additionally, fermentation conditions were optimized and scaled-up cultivation were investigated. The highest 3-HP titer was observed at 83.8 g/L with a high conversion ratio of 54% on substrate glycerol. Furthermore, a flux distribution model of glycerol metabolism in K. pneumoniae was proposed based on in silico analysis. To our knowledge, this is the highest 3-HP production in K. pneumoniae. This work has significantly advanced biological production of 3-HP from renewable carbon sources. PMID:27230116

A histamine-producing strain of Klebsiellapneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests. PMID:373626

We previously described the cloning of genes related to drug resistance from Klebsiellapneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces. PMID:25807080

Exposure to whole-body irradiation is associated with fatal gram-negative sepsis. The effect of oral therapy with three quinolones, pefloxacin, ciprofloxacin, and ofloxacin, for orally acquired Klebsiellapneumoniae infection was tested in B6D2F1 mice exposed to 8.0 Gy whole-body irradiation from bilaterally positioned 60Co sources. A dose of 10(8) organisms was given orally 2 days after irradiation, and therapy was started 1 day later. Quinolones reduced colonization of the ileum with K. pneumoniae: 16 of 28 (57%) untreated mice harbored the organisms, compared to only 12 of 90 (13%) mice treated with quinolones (P less than 0.005). K. pneumoniae was isolated from the livers of 6 of 28 untreated mice, compared to only 1 of 90 treated mice (P less than 0.001). Only 5 of 20 (25%) untreated mice survived for at least 30 days compared with 17 of 20 (85%) mice treated with ofloxacin, 15 of 20 (75%) mice treated with pefloxacin, and 14 of 20 (70%) treated with ciprofloxacin (P less than 0.05). These data illustrate the efficacy of quinolones for oral therapy of orally acquired K. pneumoniae infection in irradiated hosts.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. Previous studies demonstrated that engineered Escherichia coli strains can produce P3HP with supplementation of expensive vitamin B12. The present study examined the production of P3HP from glycerol in the recombinant Klebsiellapneumoniae strain, which naturally synthesizes vitamin B12. The genes glycerol dehydratase and its reactivation factor (dhaB123, gdrA, and gdrB from K. pneumoniae), aldehyde dehydrogenase (aldH from E. coli) were cloned and expressed in K. pneumoniae to produce 3-hydroxypropionate (3HP), with 2 genes (dhaT and yqhD) for biosynthesis of 1,3-propanediol were deleted. To obtain P3HP production, propionyl-CoA synthetase (prpE from E. coli) and polyhydroxyalkanoate synthase (phaC from Ralstonia eutropha) were introduced. Under the appropriate aeration condition, the cell yield and P3HP content were 0.24 g/L and 12.7% (wt/wt [cell dry weight]) respectively along with 2.03 g/L 3HP after 48 h cultivation. Although the yield is relatively low, this study shows the feasibility of producing P3HP in K. pneumoniae from glycerol without vitamin B12 for the first time. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of P3HP. PMID:25621933

A sample of caecal effluent was obtained from a female patient who had undergone a routine colonoscopic examination. Bacteria were isolated anaerobically from the sample, and screened against the remaining filtered caecal effluent in an attempt to isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolated on a strain identified as Klebsiellapneumoniae subsp. pneumoniae (capsular type K2, rmpA+). This Siphoviridae phage presents a rosette-like tail tip and exhibits depolymerase activity, as demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1 was shown to infect and lyse capsular type K2 strains, though it did not exhibit depolymerase activity on such hosts. The genome of KLPN1 was determined to be 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which 23 represented genes associated with structure, host recognition, packaging, DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1 (GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank: KP658157) were found to be two new members of the genus “Kp36likevirus.” PMID:26246963

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. Previous studies demonstrated that engineered Escherichia coli strains can produce P3HP with supplementation of expensive vitamin B12. The present study examined the production of P3HP from glycerol in the recombinant Klebsiellapneumoniae strain, which naturally synthesizes vitamin B12. The genes glycerol dehydratase and its reactivation factor (dhaB123, gdrA, and gdrB from K. pneumoniae), aldehyde dehydrogenase (aldH from E. coli) were cloned and expressed in K. pneumoniae to produce 3-hydroxypropionate (3HP), with 2 genes (dhaT and yqhD) for biosynthesis of 1,3-propanediol were deleted. To obtain P3HP production, propionyl-CoA synthetase (prpE from E. coli) and polyhydroxyalkanoate synthase (phaC from Ralstonia eutropha) were introduced. Under the appropriate aeration condition, the cell yield and P3HP content were 0.24 g/L and 12.7% (wt/wt [cell dry weight]) respectively along with 2.03 g/L 3HP after 48 h cultivation. Although the yield is relatively low, this study shows the feasibility of producing P3HP in K. pneumoniae from glycerol without vitamin B12 for the first time. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of P3HP. PMID:25621933

In order to detect the in vitro activity of tigecycline against multiple resistant gram-negative bacilli isolated in our hospital, tigecycline susceptibilities of clinical isolates of multiple and/or panresistant 100 Acinetobacter baumannii isolates, and 38 carbapenem resistant Klebsiellapneumoniae (17 of which were panresistant), obtained between January 2005 and August 2007, were evaluated by using E-test (AB Biodisc, Sweden). Carbapenem resistance rate was found to be 59% for A.baumannii, using Vitek2 Compact System (Bio-Merieux, France) which is present in our laboratory for routine use. Minimal inhibitory concentration (MIC) levels for tigecycline were < or =2 mcg/ml in 93% of the isolates while the MIC level was 3 mcg/ml for 7% of the isolates. Tigecycline MIC50 and MIC 90 values were 1.5 and 2 mcg/ml, respectively. Among K. pneumoniae the least resistance was detected against amikacin (52.6% resistant) while tigecycline MIC levels were between 0.13 mcg/ml and 2 mcg/ml. All of the K.pneumoniae strains were susceptible to tigecycline, and the MIC50 ve MIC90 values of these isolates were 1 mcg/ml and 1.5 mcg/ml, respectively. The in vitro susceptibility rates of tigecycline against multiple and/or panresistant A. baumannii and K. pneumoniae isolates are found to be promising for use in therapy. PMID:18697418

The management of infections due to Klebsiellapneumoniae has been complicated by the emergence of antimicrobial resistance, especially to carbapenems. Resistance to carbapenems in K. pneumoniae involves multiple mechanisms, including the production of carbapenemases (e.g., KPC, NDM, VIM, OXA-48-like), as well as alterations in outer membrane permeability mediated by the loss of porins and the upregulation of efflux systems. The latter two mechanisms are often combined with high levels of other types of β-lactamases (e.g., AmpC). K. pneumoniae sequence type 258 (ST258) emerged during the early to mid-2000s as an important human pathogen and has spread extensively throughout the world. ST258 comprises two distinct lineages, namely, clades I and II, and it seems that ST258 is a hybrid clone that was created by a large recombination event between ST11 and ST442. Incompatibility group F plasmids with blaKPC have contributed significantly to the success of ST258. The optimal treatment of infections due to carbapenemase-producing K. pneumoniae remains unknown. Some newer agents show promise for treating infections due to KPC producers; however, effective options for the treatment of NDM producers remain elusive. PMID:26169401

Klebsiellapneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multidrug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and in genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing (RNA-Seq) technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 h. ORFs upregulated with carbapenem treatment included genes involved in resistance, as well as those coding for antiporters and autoinducers. ORFs downregulated included those coding for metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real-time PCR validated the general trend of some of these differentially regulated ORFs. Treatment of K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells. PMID:25583711

Background: Klebsiellapneumoniae is among the most frequently recovered etiologic agents from nosocomial infections. This opportunistic pathogen can generate a thick layer of biofilm as one of its important virulence factors, enabling the bacteria to attach to living or abiotic surfaces, which contributes to drug resistance. Objectives: The resistance of biofilm-mediated infections to effective chemotherapy has adverse effects on patient outcomes and survival. Therefore, the aim of the present study was to evaluate the biofilm-formation capacity of clinical K. pneumoniae isolates and to perform a molecular characterization using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) to determine the dominant biofilm-producing genotype. Patients and Methods: In the present study, 94 K. pneumoniae isolates were obtained from two hospitals in Tehran, Iran. Biofilm formation was assayed by a modified procedure, then ERIC-PCR was carried out. Results: The distributions of the clinical specimens used in this study were 61.7% from urine, 18.1% from wounds, 11.7% from sputum, and 8.5% from blood. Among these isolates, 33% formed fully established biofilms, 52.1% were categorized as moderately biofilm-producing, 8.5% formed weak biofilms, and 6.4% were non-biofilm-producers. Genotyping of K. pneumoniae revealed 31 different ERIC types. Biofilm-formation ability in a special ERIC type was not observed. Conclusions: Our results indicated that an enormous proportion of K. pneumoniae isolated from sputum and surgical-wound swabs produced fully established biofilms. It is reasonable to assume the existence of a relationship between the site of infection and the formation of biofilm. A high level of genetic diversity among the K. pneumoniae strains was observed. PMID:27099694

Klebsiellapneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multidrug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and in genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing (RNA-Seq) technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 h. ORFs upregulated with carbapenem treatment included genes involved in resistance, as well as those coding for antiporters and autoinducers. ORFs downregulated included those coding for metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real-time PCR validated the general trend of some of these differentially regulated ORFs. Treatment of K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells. PMID:25583711

Antibiotic-resistant strains of Klebsiellapneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiellapneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host. PMID:26666932

1. The use of pulmonary surfactant has been proposed as a vehicle for antibiotic delivery to the alveolar compartment of the lung. This study investigated survival rates of mice with a respiratory Klebsiellapneumoniae infection treated intratracheally with tobramycin using a natural exogenous surfactant preparation as vehicle. 2. At day 1 after infection, animals were injected intratracheally with 20 microliters of the following solutions: (1) a mixture of surfactant (500 micrograms) and tobramycin (250 micrograms); (2) tobramycin (250 micrograms) alone; (3) surfactant (500 micrograms) alone; and (4) NaHCO3 buffer (control, sham-treatment). A fifth group received no treatment (control). Deaths were registered every 12 h for 8 consecutive days. 3. The results show an increased survival in the group receiving the surfactant-tobramycin mixture compared to the group receiving tobramycin alone (P < 0.05), the group receiving surfactant alone (P < 0.01) and the control groups (P < 0.01). It is concluded that intratracheal instillation of surfactant-tobramycin is superior to tobramycin alone in protecting animals from death due to a respiratory Klebsiellapneumoniae infection. PMID:8937717

The global emergence of Klebsiellapneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination. PMID:25561339

This report documents emergence of New Delhi metallo-beta-lactamase (NDM-1) and Klebsiellapneumoniae carbapenemase (KPC-2) in K. pneumoniae and Enterobacter cloacae in South Africa. NDM-1 producers have not been described in South Africa, and this is the first instance that KPC producers have been identified in Africa. The two patients infected with these carbapenemase-producing bacteria demised. PMID:22116157

The inactivation of the mgrB gene, which encodes a negative-feedback regulator of the PhoPQ signaling system, was recently shown to be a common mutational mechanism responsible for acquired polymyxin resistance among carbapenemase-producing Klebsiellapneumoniae strains from clinical sources. In this work, we show that mgrB mutants can easily be selected in vitro from different K. pneumoniae lineages, and mgrB inactivation is not associated with a significant biological cost. PMID:25691629

Increased incidence of infections due to Klebsiellapneumoniae carbapenemase (KPC)-producing Klebsiellapneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak. PMID:26637170

We report on the first TDPAC-measurements of the nuclear quadrupole interaction (NQI) of (NQI) of99Mo(β-)99Tc in the nitrogenase of the bacteria Klebsiellapneumoniae. Because nitrogenase is the only Mo-containing enzyme in Klebsiellapneumoniae under the chosen conditions, no further isolation of this enzyme was necessary. The majority of the incorporated99Mo is subjected to a well defined NQI with ω=365(7) Mrad/s, η=1 and a reorientational correlation time of τcoττ≈10nsec and is attributed to the active site of the FeMo cofactor. During sample preparation we noted a pronounced affinity of the bacteria to99mTc.

Competition and cooperation phenomena occur within highly interactive biofilm communities and several non-biocides molecules produced by microorganisms have been described as impairing biofilm formation. In this study, we investigated the anti-biofilm capacities of an ubiquitous and biofilm producing bacterium, Klebsiellapneumoniae. Cell-free supernatant from K. pneumoniae planktonic cultures showed anti-biofilm effects on most Gram positive bacteria tested but also encompassed some Gram negative bacilli. The anti-biofilm non-bactericidal activity was further investigated on Staphylococcus epidermidis, by determining the biofilm biomass, microscopic observations and agglutination measurement through a magnetic bead-mediated agglutination test. Cell-free extracts from K. pneumoniae biofilm (supernatant and acellular matrix) also showed an influence, although to a lesser extend. Chemical analyses indicated that the active molecule was a high molecular weight polysaccharide composed of five monosaccharides: galactose, glucose, rhamnose, glucuronic acid and glucosamine and the main following sugar linkage residues [→2)-α-l-Rhap-(1→]; [→4)-α-l-Rhap-(1→]; [α-d-Galp-(1→]; [→2,3)-α-d-Galp-(1→]; [→3)-β-d-Galp-(1→] and, [→4)-β-d-GlcAp-(1→]. Characterization of this molecule indicated that this component was more likely capsular polysaccharide (CPS) and precoating of abiotic surfaces with CPS extracts from different serotypes impaired the bacteria-surface interactions. Thus the CPS of Klebsiella would exhibit a pleiotropic activity during biofilm formation, both stimulating the initial adhesion and maturation steps as previously described, but also repelling potential competitors. PMID:24932475

Three Klebsiellapneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all β-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K. pneumoniae isolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred to Escherichia coli strain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, ISKpn23, the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was named blaBKC-1. Brazilian Klebsiella carbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a β-lactamase of Sinorhizobium meliloti, an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very low kcat values (0.0016 to 0.031 s−1). In fact, oxacillin was the best substrate for BKC-1 (kcat/Km, 53,522.6 mM−1 s−1). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of β-lactamases in K. pneumoniae clinical isolates. PMID:26055384

Background Knowledge of the etiology of pyogenic liver and pancreatic abscesses is an important factor in determining the success of combined surgical and antibiotic treatment. Literature shows geographical variations in the prevalence and distribution of causative organisms, and the spread of Klebsiellapneumoniae carbapenemase-producing bacteria is an emerging cause of abdominal infections. Case presentation We herein describe two cases of intra-abdominal abscesses due to monomicrobial infection by Klebsiellapneumoniae Sequence Type 258 producing K. pneumoniae carbapenemase 3 (KPC-Kp). In case 1, a 50-year-old HIV-negative Italian woman with chronic pancreatitis showed infection of a pancreatic pseudocystic lesion caused by KPC-Kp. In case 2, a 64-year-old HIV- negative Italian woman with pancreatic neoplasm and liver metastases developed a liver abscess due to KPC after surgery. Both women were admitted to our hospital but to different surgical units. The clonal relationship between the two isolates was investigated by pulsed-field gel electrophoresis (PFGE). In case 2, the patient was already colonized at admission and inter-hospital transmission of the pathogen was presumed. A long-term combination regimen of colistin with tigecycline and percutaneous drainage resulted in full recovery and clearance of the multidrug-resistant (MDR) pathogen. Conclusions Timely microbiological diagnosis, the combined use of new and old antibiotics and radiological intervention appeared to be valuable in managing these serious conditions. The emergence and dissemination of MDR organisms is posing an increasing challenge for physicians to develop new therapeutic strategies and control and prevention frameworks. PMID:21961811

Emergent hypermucoviscosity (HMV) phenotypes of Klebsiellapneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals. PMID:26951091

Three Klebsiellapneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all β-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K. pneumoniae isolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred to Escherichia coli strain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, ISKpn23, the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was named bla BKC-1. Brazilian Klebsiella carbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a β-lactamase of Sinorhizobium meliloti, an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very low kcat values (0.0016 to 0.031 s(-1)). In fact, oxacillin was the best substrate for BKC-1 (kcat /Km , 53,522.6 mM(-1) s(-1)). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of β-lactamases in K. pneumoniae clinical isolates. PMID:26055384

The purpose of this study was to investigate the clinical characteristics of hypervirulent K. pneumoniae (hvKP) induced ventilator-associated pneumonia (VAP) and the microbiological characteristics and epidemiology of the hvKP strains. A retrospective study of 49 mechanically ventilated patients with K. pneumoniae induced VAP was conducted at a university hospital in China from January 2014 to December 2014. Clinical characteristics and K. pneumoniae antimicrobial susceptibility and biofilm formation were analyzed. Genes of capsular serotypes K1, K2, K5, K20, K54 and K57 and virulence factors plasmid rmpA(p-rmpA), iroB, iucA, mrkD, entB, iutA, ybtS, kfu and allS were also evaluated. Multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) analyses were used to study the clonal relationship of the K. pneumoniae strains. Strains possessed p-rmpA and iroB and iucA were defined as hvKP. Of 49 patients, 14 patients (28.6 %) were infected by hvKP. Antimicrobial resistant rate was significantly higher in cKP than that in hvKP. One ST29 K54 extended-spectrum-beta-lactamase (ESBL) producing hvKP strain was detected. The prevalence of K1 and K2 in hvKP was 42.9 % and 21.4 %, respectively. The incidences of K1, K2, K20, p-rmpA, iroB, iucA, iutA, Kfu and alls were significantly higher in hvKP than those in cKP. ST23 was dominant among hvKP strains, and all the ST23 strains had identical RAPD pattern. hvKP has become a common pathogen of VAP in mechanically ventilated patients in China. Clinicians should increase awareness of hvKP induced VAP and enhance epidemiologic surveillance. PMID:26753990

Klebsiellapneumoniae sequence type (ST) 307, carrying blaKPC-3, blaCTX-M-15, blaOXA-1, aac(6′)-Ib-cr, and qnrB1 genes, is replacing the predominant hyperepidemic ST258 clone in Italy. Whole-genome and complete plasmid sequencing of one ST307 strain was performed and new features were identified. PMID:27056222

Klebsiella pneumoniae CICC10011, a promising 2,3-butanediol producer, has received much attention because of its high productivity. Here, the first draft genome sequence of this efficient strain may provide the genetic basis for further insights into the metabolic and regulatory mechanisms underlying the production of 2,3-butanediol at a high titer. PMID:26205860

Here, we report the genome sequence of a blaNDM-1-positive Klebsiellapneumoniae AATZP isolate cultured from a perirectal surveillance swab collected upon admission of a patient to the NIH Clinical Center in 2014. Genome sequencing of this isolate revealed three plasmids, including one carrying the blaNDM-1 gene encoding resistance to carbapenems. PMID:27417839

Urinary tract infections (UTIs) are the most frequent infections among kidney transplant (KT) patients. This case documents the emergence of New Delhi metallo-beta-lactamase (NDM-1) Klebsiellapneumonia--a factor of recurrent post-KT UTI, leading to graft loss. Spreading globally, and multidrug resistant, NDM-1 may become a great threat to transplant patients all over the world. PMID:25242796

The TEM-107 extended-spectrum β-lactamase detected in a Klebsiellapneumoniae clinical isolate had a Gly238Ser substitution compared to the TEM-43 β-lactamase. The MIC of ceftazidime was higher (64 μg/ml) than that of cefotaxime (2 μg/ml) for the isolate. Clavulanic acid reduced the MIC of ceftazidime 64-fold. PMID:21911565

This report describes a case of Clostridium difficile ribotype 033 colitis in a patient treated with multiple antibiotics for KPC-producing Klebsiellapneumoniae pancreatitis. Diagnostic, clinical and therapeutic features are discussed. To the best of our knowledge, this is the first case of C. difficile ribotype 033 clinical infection reported from Italy. PMID:27602425

We report here the draft genome sequence of uropathogenic Klebsiellapneumoniae sequence type 14 strain of serotype K2 possessing blaNDM-5, isolated from a 65-year-old male in China without a history of travel abroad. PMID:26988061

An outbreak of carbapenem-resistant Klebsiellapneumoniae strains emerged at a hospital, and was tracked in order to understand the spread of these infectious pathogens. A total of 66 K. pneumoniae strains were collected from sterile samples in 2012. The MICs of 20 antimicrobial agents were determined for all strains. Molecular typing was performed with pulsed-field gel electrophoresis (PFGE). Twelve blaKPC-producing K. pneumoniae strains isolated from ten patients were selected for whole genome sequencing. Phylogenetic reconstruction of these 12 strains was performed by the use of single-nucleotide polymorphism (SNP) row sequences of each draft genome sequence. Plasmids from the 12 strains were separated by S1 digestion and PFGE. The 12 K. pneumoniae strains isolated from the ten patients were deemed to be representative of the hospital outbreak, owing to their similar PFGE patterns. These 12 blaKPC-producing strains conferred multidrug resistance, which contrasted with the remaining 54, more susceptible, strains in the hospital. Differences in SNPs between each draft genome of the blaKPC-producing strains partitioned the 12 outbreak strains into three separate clades. The patients with each clade shared close hospital units. All 12 strains harboured at least one multidrug resistance plasmid. Strains showing high-level resistance may facilitate nosocomial dissemination and result in an infectious pathogen outbreak. Although the 12 blaKPC-producing K. pneumoniae strains possessed similar PFGE patterns, SNP variations throughout the genome allowed the strains to be divided into three clades. These results suggest that three independent transmission events led to hospital-wide dissemination of the outbreak strains. PMID:26166545

Extensively drug-resistant (XDR) Klebsiellapneumoniae is an emerging pathogen in Singapore. With limited therapeutic options available, combination antibiotics may be the only viable option. In this study, we aimed to elucidate effective antibiotic combinations against XDR K. pneumoniae isolates. Six NDM-1-producing and two OXA-181-producing K. pneumoniae strains were exposed to 12 antibiotics alone and in combination via time-kill studies. A hollow-fiber infection model (HFIM) with pharmacokinetic validation was used to simulate clinically relevant tigecycline-plus-meropenem dosing regimens against 2 XDR K. pneumoniae isolates over 240 h. The emergence of resistance against tigecycline was quantified using drug-free and selective (tigecycline at 3× the MIC) media. The in vitro growth rates were determined and serial passages on drug-free and selective media were carried out on resistant isolates obtained at 240 h. Both the polymyxin B and tigecycline MICs ranged from 1 to 4 mg/liter. In single time-kill studies, all antibiotics alone demonstrated regrowth at 24 h, except for polymyxin B against 2 isolates. Tigecycline plus meropenem was found to be bactericidal in 50% of the isolates. For the isolates that produced OXA-181-like carbapenemases, none of the 55 tested antibiotic combinations was bactericidal. Against 2 isolates in the HFIM, tigecycline plus meropenem achieved a >90% reduction in bacterial burden for 96 h before regrowth was observed until 109 CFU/ml at 240 h. Phenotypically stable and resistant isolates, which were recovered from tigecycline-supplemented plates post-HFIM studies, had lower growth rates than those of their respective parent isolates, possibly implying a substantial biofitness deficit in this population. We found that tigecycline plus meropenem may be a potential antibiotic combination for XDR K. pneumoniae infections, but its efficacy was strain specific. PMID:25691628

Inhaling concomitants from Asian sand dust (ASD) may result in exacerbation of pneumonia by the pathogen. The exacerbating effect of ASD on pneumonia induced by Klebsiellapneumoniae (KP) was investigated in ICR mice. The organic substances adsorbed onto ASD collected from the atmosphere of Iki-island in Japan were excluded by heat treatment at 360 °C for 30 min. ICR mice were instilled intratracheally with ASD at doses of 0.05 mg or 0.2 mg/mouse four times at 2-week intervals (total dose of 0.2 mg or 0.8 mg/mouse) and were administrated with ASD in the presence or absence of KP at the last intratracheal instillation. Pathologically, ASD caused exacerbation of pneumonia by KP as shown by increased inflammatory cells within the bronchiolar and the alveolar compartments. ASD enhanced the neutrophil number dose dependently as well as the expression of cytokines (IL-1β, IL-6, IL-12, IFN-γ, TNF-α) and chemokines (KC, MCP-1, MIP-1α) related to KP in BALF. In an in vitro study using RAW264.7 cells, combined treatment of ASD and KP increased gene expression of IL-1β, IL-6, IFN-β, KC, MCP-1, and MIP-1α. The same treatment tended to increase the protein level of IL-1β, TNF-α and MCP-1 in a culture medium compared to each treatment alone. The combined treatment tended to increase the gene expression of Toll-like receptor 2 (TLR2), and NALP3, ASC and caspase-1 compared with KP alone. These results suggest that the exacerbation of pneumonia by ASD + KP was due to the enhanced production of pro-inflammatory mediators via activation of TLR2 and NALP3 inflammasome pathways in alveolar macrophages.

Background β-lactamase production and porin decrease are the well-recognized mechanisms of acquired ß-lactam resistance in Klebsiellapneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and succeptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this β-lactam phenotype was the aim of this study. Methodology/Findings MICs of 9 β-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other β-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other β-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of β-lactams. Conclusion This is the first study demonstrating that efflux mechanism plays a key role in the β-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in ß-lactam resistance is specially underestimated in clinical isolates. PMID:19279676

Recently, the newly emerged hypervirulent Klebsiellapneumoniae strain (hvKP) has caused great concern globally, but the clinical features and molecular characteristics of bacteremia caused by hvKP are rarely reported in mainland China. Seventy patients with K. pneumoniae bacteremia were investigated to study the clinical features of hvKP infection from 2008 till 2012 in Beijing Chao-Yang Hospital. The molecular characteristics of the hvKP strains were also studied using PCR, multilocus sequence typing, and pulsed-field gel electrophoresis (PFGE) methods. hvKP was identified in 31.4% of the patients with K. pneumoniae bacteremia, which displayed 4 serotypes (K1, K2, K20, and K57). Patients with hvKP infection tended to have no underlying diseases compared to those with classic K. pneumoniae (cKP). More hvKP-positive patients (95.5%) had community-acquired infection than did cKP-infected patients (35.4%) (P < 0.001). The 30-day mortality rate was lower in hvKP-infected patients than in cKP-infected patients (4.5% compared to 16.7%). Resistance to tested antimicrobials was significantly greater in cKP- than in hvKP-infected patients. Two extended-spectrum-beta-lactamase (ESBL)-producing hvKP strains were found. Seven novel sequence types (STs) and 4 new alleles of K. pneumoniae were revealed. A strong correlation was found between two STs (ST23, ST1265) and the K1 serotype. The hvKP isolates (n = 22) had 14 different PFGE patterns, and among them 10 K1 isolates shared similar PFGE patterns. The emerging hvKP strain was prevalent in patients with severe community-acquired infections in healthy individuals in China. Identification of ESBL-producing hvKP strains in hvKP-infected patients will facilitate clinical management of hvKP infection. PMID:24982067

Background: Escherichia coli and Klebsiellapneumoniae are common human pathogens that cause a wide spectrum of infections. Antimicrobial resistance is a basic obstacle in the management of these infections which has different patterns in various regions. Objectives: In this study, the antibiotic resistance patterns and risk factors for multidrug-resistant (MDR) E. coli and K. pneumoniae were determined. Patients and Methods: In this cross-sectional study, a total of 250 isolates (134 E. coli and 116 K. pneumoniae) were collected and antimicrobial resistances to ampicillin, amoxicillin-clavulanic acid, amikacin, gentamycin, ceftriaxone, ceftazidime, ciprofloxacin and imipenem were evaluated by disc diffusion method and confirmed by E-test. Moreover, risk factors for MDR E. coli and K. pneumoniae were also detected. Results: The mean ages of the culture-positive cases of E. coli and K. pneumoniae were 33.39 ± 24.42 and 36.54 ± 24.66 years, respectively (P = 0.31); 137 (54.8%) cases were male and 113 (45.2%) were female (P = 0.53). Nineteen (14.2%) isolates of E. coli and 12 (10.3%) isolates of K. pneumoniae were sensitive to all the evaluated antibiotics. The prevalence of MDR E. coli and MDR K. pneumoniae was 50% and 46.6%, respectively (P = 0.59). The highest resistance for both strains was to ampicillin and no imipenem resistance was seen. The risk factors for MDR E. coli were admission history during the recent three months (P = 0.043) and antibiotic use in the previous month (P = 0.03); for MDR K. pneumoniae, they were admission in the pediatric ward (P = 0.016), surgical ward (P = 0.019), or gynecology ward (P = 0.12), admission duration of > seven days, and antibiotic use during the past month (P = 0.04). Conclusions: The prevalence of multidrug resistance was high compared with developed countries, and history of admission, antibiotic use, admission duration and admission wards were the risk factors for multidrug resistance. PMID:26587220

Some bacterial species can colonize humans and plants. It is almost impossible to prevent the contact of clinically pathogenic bacteria with food crops, and if they can persist there, they can reenter the human food chain and cause disease. On the leaf surface, microorganisms are exposed to a number of stress factors. It is unclear how they survive in such different environments. By increasing adhesion to diverse substrates, minimizing environmental differences, and providing protection against defence mechanisms, biofilms could provide part of the answer. Klebsiellapneumoniae subsp. pneumoniae is clinically important and also associated with fruit diseases, such as “pineapple fruit collapse.” We aimed to characterize biofilm formation and adhesion mechanisms of this species isolated from pineapple in comparison with a clinical isolate. No differences were found between the two isolates quantitatively or qualitatively. Both tested positive for capsule formation and were hydrophobic, but neither produced adherence fibres, which might account for their relatively weak adhesion compared to the positive control Staphylococcus epidermidis ATCC 35984. Both produced biofilms on glass and polystyrene, more consistently at 40°C than 35°C, confirmed by atomic force and high-vacuum scanning electron microscopy. Biofilm formation was maintained in an acidic environment, which may be relevant phytopathologically. PMID:24222755

Multidrug-resistant organisms (MDRO) are a great problem in hospitals, where thousands of people are infected daily, with the occurrence of high mortality rates, especially in infections caused by Klebsiellapneumoniae carbapenemase-producing K. pneumoniae (KPC-producing Kpn). The challenge is to find new compounds that can control KPC producing-Kpn infections. The aim of this study was to evaluate the antibiotic activity of the F3d fraction produced by the Pseudomonas aeruginosa LV strain against clinical isolates of KPC-producing Kpn. The results showed that the minimum inhibitory concentration of F3d (62.5 µg mL(-1)), containing an organic metallic compound, killed planktonic cells of KPC-producing Kpn strains after 30 min of incubation. At the same concentration, this fraction also showed an inhibitory effect against biofilm of these bacteria after 24 h of incubation. Treatment with the F3d fraction caused pronounced morphological alterations in both planktonic and biofilm cells of the bacteria. The inhibitory effect of the F3d fraction seems to be more selective for the bacteria than the host cells, indicating its potential in the development of new drugs for the treatment of infections caused by KPC-producing Kpn and other MDRO. PMID:26891742

Klebsiellapneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniaepneumonia. PMID:25971969

Klebsiellapneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniaepneumonia. PMID:25971969

Human β-defensins (hBDs) contribute to the protection of the respiratory tract against pathogens. It is reasonable to postulate that pathogens have developed countermeasures to resist them. Klebsiellapneumoniae capsule polysaccharide (CPS), but not the lipopolysaccharide O antigen, mediated resistance against hBD1 and hBD2. hBD3 was the most potent hBD against Klebsiella. We investigated the possibility that as a strategy for survival in the lung, K. pneumoniae may not activate the expression of hBDs. Infection of A549 and normal human bronchial cells with 52145-ΔwcaK2, a CPS mutant, increased the expression of hBD2 and hBD3. Neither the wild type nor the lipopolysaccharide O antigen mutant increased the expression of hBDs. In vivo, 52145-ΔwcaK2 induced higher levels of mBD4 and mBD14, possible mouse orthologues of hBD2 and hBD3, respectively, than the wild type. 52145-ΔwcaK2-dependent upregulation of hBD2 occurred via NF-κB and mitogen-activated protein kinases (MAPKs) p44/42, Jun N-terminal protein kinase (JNK)-dependent pathways. The increase in hBD3 expression was dependent on the MAPK JNK. 52145-ΔwcaK2 engaged Toll-like receptors 2 and 4 (TLR2 and TLR4) to activate hBD2, whereas hBD3 expression was dependent on NOD1. K. pneumoniae induced the expression of CYLD and MKP-1, which act as negative regulators for 52145-ΔwcaK2-induced expression of hBDs. Bacterial engagement of pattern recognition receptors induced CYLD and MKP-1, which may initiate the attenuation of proinflammatory pathways. The results of this study indicate that K. pneumoniae CPS not only protects the pathogen from the bactericidal action of defensins but also impedes their expression. These features of K. pneumoniae CPS may facilitate pathogen survival in the hostile environment of the lung. PMID:20008534

This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiellapneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by 1H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential. PMID:23887184

Dealing with carbapenem-resistant Klebsiellapneumoniae (CR-Kp) strains, which are generally pan-drug resistant, is an uphill task for health care professionals. Owing to limited therapeutic options and the possibility of development of resistance to commonly used biocides in hospital settings, CR-Kp infections pose a serious threat of emergence of a dreaded pandemic. The aim of the study was to highlight the possibility of emergence of biocide resistance among CR-Kp strains. A case study was conducted in a Super-specialty Hospital in September 2015. A 65-year-old female patient post-laparotomy was admitted to the General Intensive Care Unit of a Super-specialty Hospital. CR-Kp was isolated from the blood and mucus trap samples of this patient. Susceptibility testing of three commonly used biocides in our hospital, namely sodium hypochlorite (4% available chlorine), 5% w/v povidone iodine (0.5% w/v of available iodine) and absolute ethanol (99.9%), respectively, was carried out using K. pneumoniae ATCC 700603 as control. The test isolate showed reduced susceptibility to sodium hypochlorite in comparison to K. pneumoniae ATCC 700603. The possibility of emergence of biocide resistance among CR-Kp strains poses a threat of disrupting our ongoing efforts for implementation of effective infection control measures. PMID:27514961

The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiellapneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens. PMID:25163721

Background. While the importance of fluid dynamical conditions is well recognized in the growth of biofilms, their role during bacteremia is unknown. We examined the impact of physiological fluid shear forces on the development of multicellular aggregates of Klebsiellapneumoniae. Methods. Wild-type and O-antigen or capsular mutants of K. pneumoniae were grown as broth culture in a Taylor-Couette flow cell configured to provide continuous shear forces comparable to those encountered in the human arterial circulation (ie, on the order of 1.0 Pa). The size distribution and antibiotic resistance of aggregates formed in this apparatus were determined, as was their ability to persist in the bloodstream of mice following intravenous injection. Results. Unlike growth in shaking flasks, bacteria grown in the test apparatus readily formed aggregates, a phenotype largely absent in capsular mutants and to a lesser degree in O-antigen mutants. Aggregates were found to persist in the bloodstream of mice. Importantly, organisms grown under physiological shear were found to have an antibiotic resistance phenotype intermediate between that of fully planktonic and biofilm states. Conclusions. When grown under intravascular-magnitude fluid dynamic conditions, K. pneumoniae spontaneously develops into multicellular aggregates that are capable of persisting in the circulation and exhibit increased antibiotic resistance. PMID:22711903

The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiellapneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens. PMID:25163721

Health-care-associated infections by multi-drug-resistant bacteria constitute one of the greatest challenges to modern medicine. Bacterial pathogens devise various mechanisms to withstand the activity of a wide range of antimicrobial compounds, among which the acquisition of carbapenemases is one of the most concerning. In Klebsiellapneumoniae, the dissemination of the K. pneumoniae carbapenemase is tightly connected to the global spread of certain clonal lineages. Although antibiotic resistance is a key driver for the global distribution of epidemic high-risk clones, there seem to be other adaptive traits that may explain their success. Here, we exploited the power of deep transcriptome profiling (RNA-seq) to shed light on the transcriptomic landscape of 37 clinical K. pneumoniae isolates of diverse phylogenetic origins. We identified a large set of 3346 genes which was expressed in all isolates. While the core-transcriptome profiles varied substantially between groups of different sequence types, they were more homogenous among isolates of the same sequence type. We furthermore linked the detailed information on differentially expressed genes with the clinically relevant phenotypes of biofilm formation and bacterial virulence. This allowed for the identification of a diminished expression of biofilm-specific genes within the low biofilm producing ST258 isolates as a sequence type-specific trait. PMID:26261087

The filtration in 1,3-propanediol (1,3-PD) downstream process is influenced by the large amounts of capsular polysaccharides (CPS) produced by Klebsiellapneumoniae CGMCC 1.6366. The morphological and fermentation properties were investigated with the CPS-deficient mutant K. pneumoniae CGMCC 1.6366 CPS. Similar biomass was obtained with CGMCC 1.6366, and the mutant strain in batch cultures indicating the cell growth was slightly inhibited by CPS defection. The viscosity of fermentation broth by mutant strain decreased by 27.45%. The flux with ceramic membrane filter was enhanced from 168.12 to 303.6 l h(-1) m(-2), exhibiting the great importance for downstream processing of 1,3-PD fermentation. The products spectrum of mutant isolate changed remarkably regarding to the concentration of fermentation products. The synthesis of important 1,3-PD and 2,3-butanediol was enhanced from 9.73 and 4.06 g l(-1) to 10.37 and 4.77 g l(-1) in batch cultures. The noncapsuled K. pneumoniae provided higher 1,3-PD yield of 0.54 mol mol(-1) than that of encapsuled wild parent in batch cultures. The fed-batch fermentation of mutant strain resulted in 1,3-PD concentration, yield, and productivity of 78.13 g l(-1), 0.53 mol mol(-1), and 1.95 g l(-1) h(-1), respectively. PMID:19936735

In response to molecular oxygen and/or fixed nitrogen, the product of the Klebsiellapneumoniae nitrogen fixation L (nifL) gene inhibits NifA-mediated transcriptional activation. Nitrogen regulation of NifL function occurs at two levels: transcription of the nifLA operon is regulated by the general Ntr system, and the activity of NifL is controlled by an unknown mechanism. We have studied the regulation of NifL activity in Escherichia coli and Salmonella typhimurium by monitoring its inhibition of NifA-mediated expression of a K. pneumoniae phi(nifH'-'lacZ) fusion. The activity of the NifL protein transcribed from the tac promoter is regulated well in response to changes of oxygen and/or nitrogen status, indicating that no nif- or K. pneumoniae-specific product is required. Unexpectedly, strains carrying ntrC (glnG) null alleles failed to release NifL inhibition, despite the fact that synthesis of NifL was no longer under Ntr control. Additional evidence indicated that it is indeed the transcriptional activation capacity of NtrC, rather than its repression capacity, that is needed, and hence it is a plausible hypothesis that NtrC activates transcription of a gene(s) whose product(s) in turn functions to relieve NifL inhibition under nitrogen-limiting conditions. PMID:9393710

Objective To analyze the profile of patients with microorganisms resistant to carbapenems, and the prevalence of the enzyme Klebsiellapneumoniae carbapenemase in interobacteriaceae. Methods Retrospective descriptive study. From the isolation in bacteriological tests ordered by clinicians, we described the clinical and epidemiological characteristics of patients with enterobacteria resistants to carbapenems at a university hospital, between March and October 2013. Results We included 47 isolated patients in this study, all exhibiting resistance to carbapenems, including 9 patients who were confirmed as infected/colonized with K. pneumoniae carbapenemase. Isolation in tracheal aspirates (12; 25.5%) predominated. The resistance to ertapenem, meropenem, and imipenem was 91.5%, 83.0% and 80.0%, respectively. Aminoglycosides was the class of antimicrobials that showed the highest sensitivity, 91.5% being sensitive to amikacin and 57.4% to gentamicin. Conclusion The K. pneumoniae carbapenemase was an important agent in graun isotaling in hospital intection. The limited therapeutic options emphasize the need for rapid laboratory detection, as well as the implementation of measures to prevent and control the spread of these pathogens. PMID:25295446

The polysaccharide (PS) derived from K. pneumoniae NCTC 5055 lipopolysaccharide (LPS) was covalently linked to tetanus toxoid by using carbodimide with adipic acid dihydrazide as a spacer molecule. The conjugate was found to be non-toxic and non-pyrogenic at 100 microg dose level. At a similar dose, the conjugate did not elicit any local skin reaction on intradermal preparatory injection in rabbits. The conjugate was immunoprotective as was evident from the decrease in relative colonization of bacteria in lungs of immunized rats as compared to the control animals. Immunization with the conjugate resulted in alveolar macrophage activation in terms of their ability to phagocytose bacteria in vitro. PMID:15691064

HpxW from the ubiquitous pathogen Klebsiellapneumoniae is involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed. PMID:27303801

A mutant strain of Klebsiellapneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilis pdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167. PMID:21186120

The expression of Klebsiellapneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin. PMID:8534083

Carbapenem-resistant Klebsiellapneumoniae (CRKP) is an increasing global threat. Here, we describe the prevalence and impact of tigecycline use in a cohort of patients with CRKP bacteriuria nested within a multicenter, prospective study. In the 21 month study period, 260 unique patients were included. Tigecycline was given to 80 (31%) patients. The use of tigecycline during the index hospitalization was significantly associated with the subsequent development of tigecycline resistance in the same patient (OR 6.13, 95%CI 1.15–48.65, p=0.03). In conclusion, the use of tigecycline with CRKP bacteriuria is common, and is associated with the subsequent development of tigecycline resistance. PMID:24931918

Supplementation with acetate under low levels was used as a novel approach to control the morphological development of Klebsiellapneumoniae aimed to improve 1,3-propanediol (1,3-PD) production. A full range of morphological types formed from rod shape to oval shape even round shape in response to different concentrations of acetate. The cell growth and 1,3-PD productions in the shake flasks with 0.5 g/L acetate addition were improved by 9.4 and 28.37%, respectively, as compared to the control, while the cell became shorter and began to lose its original shape. The cell membrane penetration by acetate was investigated by the biomimetic vesicles, while higher concentration of acetate led to more moderate colorimetric transitions. Moreover, the percentage composition of unsaturated fatty acid (UFA) was increased as well as the increased concentrations of acetate, whereas higher UFA percentage, higher fluidity of bacterial cell membrane. PMID:23892619

Carbapenem-resistantKlebsiella pneumoniaestrains classified as multilocus sequence type 258 (ST258) are among the most widespread multidrug-resistant hospital-acquired pathogens. Treatment of infections caused by these organisms is difficult, and mortality is high. The basis for the success of ST258, outside of antibiotic resistance, remains incompletely determined. Here we tested the hypothesis that ST258K. pneumoniaehas enhanced capacity to circumvent killing by human neutrophils, the primary cellular defense against bacterial infections. There was limited binding and uptake of ST258 by human neutrophils, and correspondingly, there was limited killing of bacteria. On the other hand, transmission electron microscopy revealed that any ingested organisms were degraded readily within neutrophil phagosomes, thus indicating that survival in the neutrophil assays is due to limited phagocytosis, rather than to microbicide resistance after uptake. Our findings suggest that enhancing neutrophil phagocytosis is a potential therapeutic approach for treatment of infection caused by carbapenem-resistant ST258K. pneumoniae. PMID:26768252

A 39-year-old woman visited the emergency room complaining of right eye pain and swelling over the preceding three days. The ophthalmologist's examination revealed orbital cellulitis and diabetic retinopathy in the right eye, although the patient had no prior diagnosis of diabetes. It was therefore suspected that she had diabetes and orbital cellulitis, and she was started on multiple antibiotic therapies initially. She then underwent computed tomography scans of the orbit and neck and magnetic resonance imaging of the brain. These studies showed an aggravated orbital cellulitis with abscess formation, associated with venous thrombophlebitis, thrombosis of the internal carotid artery, and mucosal thickening of maxillary sinus with multiple paranasal abscesses. Three days later, initial blood culture grew Klebsiellapneumoniae. She recovered after incision and drainage and antibiotic therapy for 37 days. PMID:20195414

This study shows for the first time the mechanism of carbapenem resistance of a Klebsiellapneumoniae clinical isolate TJ8 recovered from Tianjin Medical University General Hospital ,China. The modified Hodge test and EDTA synergy test were performed for the screening of carbapenemases and metallo-β-lactamases (MBLs), respectively. Polymerase chain reactions and DNA sequencing confirmed that the strain carried IMP-4 metallo-β-lactamase (MBL) , SHV-11 and TEM-1 β-lactamase. Class I integron was positive and gave a 3.0-kb PCR amplicon .IMP-4 was located in Class I integron 5’CS. The gene determinants were organized in the order of blaIMP-4-orfII-orfIII.In all, the results show that IMP-4 MBL production caused the TJ8 resistance to carbapenems. PMID:24031907

The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiellapneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

Hypervirulent strains of Klebsiellapneumoniae (hvKP) are genetic variants of K. pneumoniae which can cause life-threatening community-acquired infection in healthy individuals. Currently, methods for efficient differentiation between classic K. pneumoniae (cKP) and hvKP strains are not available, often causing delay in diagnosis and treatment of hvKP infections. To address this issue, we devised a Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) approach for rapid identification of K1 hvKP strains. Four standard algorithms, genetic algorithm (GA), support vector machine (SVM), supervised neural network (SNN), and quick classifier (QC), were tested for their power to differentiate between K1 and non-K1 strains, among which SVM was the most reliable algorithm. Analysis of the receiver operating characteristic curves of the interest peaks generated by the SVM model was found to confer highly accurate detection sensitivity and specificity, consistently producing distinguishable profiles for K1 hvKP and non-K1 strains. Of the 43 K. pneumoniae modeling strains tested by this approach, all were correctly identified as K1 hvKP and non-K1 capsule type. Of the 20 non-K1 and 17 K1 hvKP validation isolates, the accuracy of K1 hvKP and non-K1 identification was 94.1 and 90.0%, respectively, according to the SVM model. In summary, the MALDI-TOF MS approach can be applied alongside the conventional genotyping techniques to provide rapid and accurate diagnosis, and hence prompt treatment of infections caused by hvKP. PMID:26733976

Infections caused by multidrug resistant Klebsiellapneumoniae have been increasingly reported in many parts of the world. A total of 93 Malaysian multidrug resistant K. pneumoniae isolated from patients attending to University of Malaya Medical Center, Kuala Lumpur, Malaysia from 2010-2012 were investigated for antibiotic resistance determinants including extended-spectrum beta-lactamases (ESBLs), aminoglycoside and trimethoprim/sulfamethoxazole resistance genes and plasmid replicons. CTX-M-15 (91.3%) was the predominant ESBL gene detected in this study. aacC2 gene (67.7%) was the most common gene detected in aminoglycoside-resistant isolates. Trimethoprim/sulfamethoxazole resistance (90.3%) was attributed to the presence of sul1 (53.8%) and dfrA (59.1%) genes in the isolates. Multiple plasmid replicons (1-4) were detected in 95.7% of the isolates. FIIK was the dominant replicon detected together with 13 other types of plasmid replicons. Conjugative plasmids (1-3 plasmids of ~3-100 kb) were obtained from 27 of 43 K. pneumoniae isolates. An ESBL gene (either CTX-M-15, CTX-M-3 or SHV-12) was detected from each transconjugant. Co-detection with at least one of other antibiotic resistance determinants [sul1, dfrA, aacC2, aac(6ˊ)-Ib, aac(6ˊ)-Ib-cr and qnrB] was noted in most conjugative plasmids. The transconjugants were resistant to multiple antibiotics including β-lactams, gentamicin and cotrimoxazole, but not ciprofloxacin. This is the first study describing the characterization of plasmids circulating in Malaysian multidrug resistant K. pneumoniae isolates. The results of this study suggest the diffusion of highly diverse plasmids with multiple antibiotic resistance determinants among the Malaysian isolates. Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of the multidrug resistant bacteria in healthcare settings. PMID:26203651

Enzymes of the OXA-48 family have become some of the most important beta-lactamases in the world. A new OXA-48 variant (OXA-370) was first described for an Enterobacter hormaechei strain isolated in Rio Grande do Sul (southern region of Brazil) in 2013. Here we report detection of the blaOXA-370 gene in 24 isolates belonging to three Enterobacteriaceae species (22 Klebsiellapneumoniae isolates, 1 Enterobacter cloacae isolate, and 1 Enterobacter aerogenes isolate) collected from five hospitals in Rio de Janeiro, Brazil, in 2013 and 2014. The isolates showed a multidrug resistance profile, and 12.5% were resistant to polymyxin B. Besides blaOXA-370, no other carbapenemase genes were observed by PCR, whereas blaOXA-1 was found in all isolates and 22 isolates (91.6%) possessed blaCTX-M-15. Molecular typing of the K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two clonal groups, i.e., KpA (21 isolates) and KpB (1 isolate). KpA was characterized as sequence type 16 (ST16) and KpB as ST1041 by multilocus sequence typing (MLST). ST16 has been observed for KPC-producing K. pneumoniae in Rio de Janeiro. Plasmid analysis performed with six representative OXA-370-producing isolates showed plasmids harboring the blaOXA-370 gene in all strains, ranging from 25 kb to 150 kb. This study suggests that there is an urgent need to investigate the presence of OXA-370 and dissemination of the K. pneumoniae ST16 clone carrying this gene in Brazil. PMID:25987619

Carbapenem-resistant Klebsiellapneumoniae strains have emerged as a cause of life-threatening infections in susceptible individuals (e.g., transplant recipients and critically ill patients). Strains classified as multilocus sequence type (ST) 258 are among the most prominent causes of carbapenem-resistant K. pneumoniae infections worldwide, but the basis for the success of this lineage remains incompletely determined. To gain a more comprehensive view of the molecules potentially involved in the success of ST258, we used a proteomics approach to identify surface-associated and culture supernatant proteins produced by ST258. Protein samples were prepared from varied culture conditions in vitro, and were analyzed by a combination of two-dimensional electrophoresis and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We identified a total of 193 proteins in outer membrane preparations from bacteria cultured in Luria-Bertani broth (LB) or RPMI 1640 tissue culture media (RPMI). Compared with LB, several iron-acquisition proteins, including IutA, HmuR, HmuS, CirA, FepA, FitA, FoxA, FhuD, and YfeX, were more highly expressed in RPMI. Of the 177 proteins identified in spent media, only the fimbrial subunit, MrkA, was predicted to be extracellular, a finding that suggests few proteins (or a limited quantity) are freely secreted by ST258. Notably, we discovered 203 proteins not reported in previous K. pneumoniae proteome studies. In silico modeling of proteins with unknown function revealed several proteins with beta-barrel transmembrane structures typical of porins, as well as possible host-interacting proteins. Taken together, these findings contribute several new targets for the mechanistic study of drug-resistance and pathogenesis by ST258 K. pneumoniae isolates. PMID:25893665

Klebsiellapneumoniae is a Gram-negative bacillus belonging to the family Enterobacteriaceae. In the past 20 years, K. pneumoniae has become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA). The formation of biofilm facilitates bacterial colonization and has been implicated in reduced susceptibility to the host immune response. To investigate genes related to biofilm formation in a PLA-associated K. pneumoniae strain, a transposon mutant library was screened by microtiter plate assay to identify isolates impaired for biofilm formation. One of the mutants was disrupted in celB, encoding the putative cellobiose-specific subunit IIC of enzyme II (EIIC) of a carbohydrate phosphotransferase system (PTS). This transmembrane protein is responsible for recognizing and binding specific sugars and transporting them across the cell membrane into the cytoplasm. Deletion and chromosomal complementation of celB confirmed, by microtiter plate and slide culture assays, that celB was indeed responsible for biofilm formation. Cellobiose-specific PTS activities of deletion mutants grown in LB broth and 0.005% cellobiose minimal medium were markedly lower than that of the wild-type strain grown under the same conditions, thereby confirming the involvement of celB in cellobiose transport. In 0.005% cellobiose minimal medium, the celB mutant showed a delay in growth compared to the wild-type strain. In a mouse model of intragastric infection, deletion of the celB gene increased the survival rate from 12.5% to 87.5%, which suggests that the celB deletion mutant also exhibited reduced virulence. Thus, the celB locus of K. pneumoniae may contribute to biofilm formation and virulence through the metabolism of cellobiose. PMID:22566508

The growing number of Klebsiellapneumoniae infections, commonly acquired in hospitals, has drawn great concern. It has been shown that the K1 and K2 capsular serotypes are the most detrimental strains, particularly to those with diabetes. The K1 cps (capsular polysaccharide) locus in the NTUH-2044 strain of the pyogenic liver abscess (PLA) K. pneumoniae has been identified recently, but little is known about the functions of the genes therein. Here we report characterization of a group of cps genes and their roles in the pathogenesis of K1 K. pneumoniae. By sequential gene deletion, the cps gene cluster was first re-delimited between genes galF and ugd, which serve as up- and down-stream ends, respectively. Eight gene products were characterized in vitro and in vivo to be involved in the syntheses of UDP-glucose, UDP-glucuronic acid and GDP-fucose building units. Twelve genes were identified as virulence factors based on the observation that their deletion mutants became avirulent or lost K1 antigenicity. Furthermore, deletion of kp3706, kp3709 or kp3712 (ΔwcaI, ΔwcaG or Δatf, respectively), which are all involved in fucose biosynthesis, led to a broad range of transcriptional suppression for 52 upstream genes. The genes suppressed include those coding for unknown regulatory membrane proteins and six multidrug efflux system proteins, as well as proteins required for the K1 CPS biosynthesis. In support of the suppression of multidrug efflux genes, we showed that these three mutants became more sensitive to antibiotics. Taken together, the results suggest that kp3706, kp3709 or kp3712 genes are strongly related to the pathogenesis of K. pneumoniae K1. PMID:21765903

The mode of succinate mediated repression of mineral phosphate solubilization and the role of repressor in suppressing phosphate solubilization phenotype of two free-living nitrogen fixing Klebsiellapneumoniae strains was studied. Organic acid mediated mineral phosphate solubilization phenotype of oxalic acid producing Klebsiellapneumoniae SM6 and SM11 were transcriptionally repressed by IclR in presence of succinate as carbon source. Oxalic acid production and expression of genes of the glyoxylate shunt (aceBAK) was found only in glucose but not in succinate- and glucose+succinate-grown cells. IclR, repressor of aceBAK operon, was inactivated using an allelic exchange system resulting in derepressed mineral phosphate solubilization phenotype through constitutive expression of the glyoxylate shunt. Insertional inactivation of iclR resulted in increased activity of the glyoxylate shunt enzymes even in succinate-grown cells. An augmented phosphate solubilization up to 54 and 59% soluble phosphate release was attained in glucose+succinate-grown SM6Δ and SM11Δ strains respectively, compared to glucose-grown cells, whereas phosphate solubilization was absent or negligible in wildtype cells grown in glucose+succinate. Both wildtype and iclR deletion strains showed similar indole-3-acetic acid production. Wheat seeds inoculated with wildtype SM6 and SM11 improved both root and shoot length by 1.2 fold. However, iclR deletion SM6Δ and SM11Δ strains increased root and shoot length by 1.5 and 1.4 folds, respectively, compared to uninoculated controls. The repressor inactivated phosphate solubilizers better served the purpose of constitutive phosphate solubilization in pot experiments, where presence of other carbon sources (e.g., succinate) might repress mineral phosphate solubilization phenotype of wildtype strains. PMID:26381651

Enterobacteriaceae strains producing the Klebsiellapneumoniae carbapenemase (KPC) have disseminated worldwide, causing an urgent threat to public health. KPC-producing strains often exhibit low-level carbapenem resistance, which may be missed by automated clinical detection systems. In this study, eight Klebsiellapneumoniae strains with heterogeneous resistance to imipenem were used to elucidate the factors leading from imipenem susceptibility to high-level resistance as defined by clinical laboratory testing standards. Time-kill analysis with an inoculum as low as 3 × 106 CFU/ml and concentrations of imipenem 8- and 16-fold higher than the MIC resulted in the initial killing of 99.9% of the population. However, full recovery of the population occurred by 20 h of incubation in the same drug concentrations. Population profiles showed that recovery was mediated by a heteroresistant subpopulation at a frequency of 2 × 10−7 to 3 × 10−6. Samples selected 2 h after exposure to imipenem were as susceptible as the unexposed parental strain and produced the major outer membrane porin OmpK36. However, between 4 to 8 h after exposure, OmpK36 became absent, and the imipenem MIC increased at least 32-fold. Individual colonies isolated from cultures after 20 h of exposure revealed both susceptible and resistant subpopulations. Once induced, however, the high-level imipenem resistance was maintained, and OmpK36 remained unexpressed even without continued carbapenem exposure. This study demonstrates the essential coordination between blaKPC and ompK36 expression mediating high-level imipenem resistance from a population of bacteria that initially exhibits a carbapenem-susceptibility phenotype. PMID:25801565

Carbapenemases are increasingly important antimicrobial resistance determinants. Little is known about the carbapenem resistance mechanisms in Sri Lanka. We examined 22 carbapenem-resistant Klebsiellapneumoniae from Sri Lanka to determine their β-lactam resistance mechanisms. The predominant resistance mechanisms we detected in this study were OXA-181, NDM-1 carbapenemases and extended-spectrum β-lactamase CTX-M-15. All isolates were then genotyped by pulsed-field gel electrophoresis, variable-number tandem repeat sequence analysis and multilocus sequence typing, and seven distinct genotypes were observed. Five OXA-181-positive Klebsiellapneumoniae isolates were genotypically related to an isolate of Indian origin. Multilocus sequence typing found that these related isolates belong to ST-14, which has been associated with dissemination of OXA-181 from the Indian subcontinent. Other genotypes we discovered were ST-147 and ST-340, also associated with intercontinental spread of carbapenemases of suspected subcontinental origin. The major porin genes ompK35 and ompK36 from these isolates had insertions, deletions and substitutions. Some of these were exclusive to strains within single pulsotypes. We detected one ompK36 variant, ins AA134-135GD, in six ST-14- and six ST-147, blaOXA-181-positive isolates. This porin mutation was an independent predictor of high-level meropenem resistance in our entire Sri Lankan isolate collection (P=0.0030). Analysis of the Sri Lankan ST-14 and ST-147 ins AA134-135GD-positive isolates found ST-14 was more resistant to meropenem than other isolates (mean MIC: 32±0 µg ml(-1) and 20±9.47 µg ml(-1), respectively, P=0.0277). The likely international transmission of these carbapenem resistance determinants highlights the need for regional collaboration and prospective surveillance of carbapenem-resistant Enterobacteriaceae. PMID:24855071

Purpose Tigecycline is one of the drugs used to treat multi-drug resistant Klebsiellapneumoniae (K. pneumoniae) infections, including complicated skin and soft tissue infections, complicated intra-abdominal infection, and community-acquired pneumonia in the Republic of Korea. However, since its commercial release, K. pneumoniae resistance against tigecycline has been reported, and there is a serious concern about the spread of tigecycline resistant bacteria. Materials and Methods In this study, we collected and analyzed 342 isolates from 23 hospitals in the Republic of Korea to determine the mechanisms of tigecycline susceptibility and their clonal types. The hospitals include several from each province in the Republic of Korea, except Jeju, an island province, and nonsusceptibility among the isolates was tested by the disk diffusion method. In our lab, susceptibility was checked again using the broth dilution method, and clonal types were determined using the multilocus sequence typing protocol. Real-time PCR was performed to measure the ramR mutation in the isolates nonsusceptible to tigecycline, which would suggest an increased expression of the AcrAB multidrug pump. Results Fifty-six K. pneumoniae isolates were found to be nonsusceptible, 16% of the 342 collected. Twenty-seven and nine isolates of the tigecycline nonsusceptible isolates had mutations in the ramR and rpsJ genes, respectively; while 18 nonsusceptible isolates harbored the tetA gene. Comparison of isolates with and without ramR mutation showed a significant statistical difference (p<0.05) for expression of AcrAB. Moreover, the most common clonal types, as observed in our study, appear to be ST11 and ST789. Conclusion Several dominate clonal types infer tigecycline resistance to K. pneumoniae, including ST11, ST768, ST15, ST23, ST48, and ST307. There does not seem to be a transferrable medium, such as plasmid, for the resistance yet, although mutation of the ramR gene may be a common event

Introduction: Gram negative organisms are one of the major causes of nosocomial diseases. Development of resistance to antibiotics by these organisms increases their risk in clinical treatment of patients. It also affects morbidity and mortality hence needs to be monitored and controlled. Aim: The aim of the present study was to analyse the correlation between consumption of parenteral antibiotics and the rates of antimicrobial resistance among the Escherichia coli and Klebsiellapneumoniae isolates collected during Dec 2010 - Jun 2013 from JIPMER hospital. Materials and Methods: Consumption data of parenteral antibiotics in J01 category of Anatomical Therapeutic Chemical (ATC) in JIPMER was obtained and expressed in Defined Daily Doses (DDD) per 1000 inhabitants. Valid consumption and resistance data during the period Dec 2010 to Jun 2013 were obtained at 6 month intervals and were correlated to draw a relationship between antimicrobial consumption and its impact on drug resistance for Escherichia coli and Klebsiellapneumoniae. Results: Escherichia coli isolates showed high resistance for increased use of gentamycin and ciprofloxacin. Increase in antibiotic consumption increases the resistance for Escherichia coli except for amikacin. Among the Klebsiella isolates, meropenem and gentamycin showed high correlations followed by ceftazidime, amikacin, ceftriaxone and ciprofloxacin. Conclusion: In summary, a statistically significant association was noticed between consumption of the studied antimicrobials and resistance of Escherichia coli isolates, except for amikacin and ceftazidime. In the case of Klebsiellapneumoniae, there was a statistically significant association between the resistance rates and consumption of gentamycin, ceftazidime and meropenem. Further, a linear relationship was noted between antimicrobial consumption and resistant isolates of Escherichia coli and Klebsiellapneumoniae, except for Escherichia coli resistance to amikacin. PMID:25859453

Four hundred and eighty-nine independent Nif- strains containing 260 point, 130 millimicron-induced, and 99 deletion mutations in nif in the Klebsiellapneumoniae chromosome were isolated. Three hundred and ninety insertion and point mutations were mapped with millimicron-induced deletions carried on 44 plasmids derived from pTM4010, a recombinant R factor containing the his-nif region of K. pneumoniae. The 99 chromosomal deletions in the nif region were mapped with 69 derivatives of pTM4010 carrying insertion and point mutations in nif. Complementation analysis between 84 derivatives of pTM4010 carrying nif mutations and Rec- derivatives of the 390 Nif- mutants identified 14 genes. The nif mutations were ordered into 49 deletion groups with a gene order of his...nifQBALFMVSNEKDHJ. Complementation analysis of millimicron-induced, amber, frameshift, and deletion mutations indicates there are five polycistronic and two monocistronic operons: nifQ nifB, nifA nifL, nifF, nifM nifV nifS, nifN nifE, nifK nifD nifH, and nifJ. Transcription is from right to left in all polycistronic operons. PMID:361693

Plasmid-dependent overexpression of enzyme(s) aims to divert carbon flux toward a desired compound. One drawback of this strategy is compromise of growth due to massive consumption of host resources. Here we show that replenishment of sigma factor rpoE improves the growth of Klebsiellapneumoniae. The gene rpoE was expressed alone or coexpressed with Ald4 (an aldehyde dehydrogenase from Saccharomyces cerevisiae) in K. pneumoniae. We found that the Ald4 activity was higher in the strain coexpressing Ald4 and rpoE (32.3 U/mg) than that expressing Ald4 alone (29.9 U/mg). Additionally, under shake-flask conditions, the strain coexpressing Ald4 and rpoE produced 0.5 g 3-hydroxypropionic acid (3-HP) and 9.8 g 1,3-propanediol (1,3-PD) per liter in 24 h, which were 1.6- and 0.85-fold enhancement, respectively, compared to those expressing Ald4 alone. Notably, under non-optimized bioreactor conditions, the strain coexpressing Ald4 and rpoE produced 13.5 g 3-HP and 37.8 g 1,3-PD per liter with glycerol conversion ratio of 0.45 mol/mol. These results indicate that replenishment of rpoE enhanced promoter activity and stimulated glycerol consumption. PMID:27570311

Fourteen clinical isolates of Klebsiellapneumoniae with extended-spectrum beta-lactamases (ESBLs) were detected by the double disk synergy test and the Etest ESBL strip. Co-resistances included high MICs for aminoglycosides, fluoroquinolones, tetracyclines, and trimethoprim/sulphamethoxazole. Co-resistance was not observed in five of the 14 strains. These isolates were all genetically distinct as determined by the automated ribotyping method. Isoelectric focusing documented the presence of multiple beta-lactamases (one to four per isolate) with pIs ranging from 5.4 to 8.4. The majority of isolates contained beta-lactamases with pI values of 7.6 and 8.4 consistent with SHV-type ESBLs and an Amp C enzyme, respectively. Emerging ESBL strains in K. pneumoniae compromise the use of agents such as cefotaxime, ceftriaxone, ceftazidime in China; leading to the expansion of quality infection control practices and formulary management programmes to minimize clonal expansion. PMID:11516943

Previous studies have implicated glutamine synthetase (L-glutamate:ammonia ligase [adenosine diphosphate for-ing], EC 6.6.1.2) as a major controlling element of the nitrogen fixation (nif) genes in Klebsiellapneumoniae. We report here the isolation of a new class of K. pneumoniae mutants which exhibit altered patterns of nif and hut (histidine utlization) regulation. The expression of nif in these mutants, which were isolated as Gln+ (glutamine nonrequiring) revertants of a particular glnA mutation, is extremely sensitive to ammonia repression. These mutants have a Nif- Hut- phenotype at external ammonia concentrations at which wild-type strains are Nif+ Hut+. On the other hand, these mutants can be fully derepressed for nif at very low ammonia concentrations. We adopted the nomenclature "GlnR- (Nif- Hut-)" to facilitate discussion of the phenotype of these mutant strains. The mutations in these strains which confer the GlnR- phenotype map at or near glnA, the structural gene for glutamine synthetase. PMID:40960

Klebsiellapneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilation pathway. We previously identified structural genes for assimilatory nitrate and nitrite reductases, nasA and nasB, respectively. We report here our further identification of four genes, nasFEDC, upstream of the nasBA genes. The nasFEDCBA genes probably form an operon. Mutational and complementation analyses indicated that both the nasC and nasA genes are required for nitrate assimilation. The predicted NASC protein is homologous to a variety of NADH-dependent oxidoreductases. Thus, the NASC protein probably mediates electron transfer from NADH to the NASA protein, which contains the active site for nitrate reduction. The deduced NASF, NASE, and NASD proteins are homologous to the NRTA, NRTB, and NRTD proteins, respectively, that are involved in nitrate uptake in Synechococcus sp. (T. Omata, X. Andriesse, and A. Hirano, Mol. Gen. Genet. 236:193-202, 1993). Mutational and complementation studies indicated that the nasD gene is required for nitrate but not nitrite assimilation. By analogy with the Synechococcus nrt genes, we propose that the nasFED genes are involved in nitrate transport in K. pneumoniae. PMID:8169203

Klebsiellapneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilation pathway. We previously identified structural genes for assimilatory nitrate and nitrite reductases, nasA and nasB, respectively. We report here our further identification of four genes, nasFEDC, upstream of the nasBA genes. The nasFEDCBA genes probably form an operon. Mutational and complementation analyses indicated that both the nasC and nasA genes are required for nitrate assimilation. The predicted NASC protein is homologous to a variety of NADH-dependent oxidoreductases. Thus, the NASC protein probably mediates electron transfer from NADH to the NASA protein, which contains the active site for nitrate reduction. The deduced NASF, NASE, and NASD proteins are homologous to the NRTA, NRTB, and NRTD proteins, respectively, that are involved in nitrate uptake in Synechococcus sp. (T. Omata, X. Andriesse, and A. Hirano, Mol. Gen. Genet. 236:193-202, 1993). Mutational and complementation studies indicated that the nasD gene is required for nitrate but not nitrite assimilation. By analogy with the Synechococcus nrt genes, we propose that the nasFED genes are involved in nitrate transport in K. pneumoniae. PMID:8169203

We evaluated the antimicrobial activity and some mechanisms used by subinhibitory and inhibitory concentrations of the essential oil, obtained from leaves of Plectranthus amboinicus, against a standard strain of Klebsiellapneumoniae and 5 multiresistant clinical isolates of the bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), the rate of kill and the pH sensitivity of the essential oil were determined by microdilution tests performed in 96-well plates. Subinhibitory and inhibitory concentrations of the essential oil were tested in order to check its action on K. pneumoniae membrane permeability, capsule expression, urease activity and cell morphology. The MIC and MBC of the essential oil were 0.09±0.01%. A complete inhibition of the bacterial growth was observed after 2 h of incubation with twice the MIC of the essential oil. A better MIC was found when neutral or alkaline pH broth was used. Alteration in membrane permeability was found by the increase of crystal violet uptake when the bacteria were incubated with twice the MIC levels of the essential oil. The urease activity could be prevented when all the subinhibitory concentrations were tested in comparison to the untreated group (p<0.001). Alteration of the bacterial morphology besides inhibition of the capsule expression was verified by atomic force microscopy, and Anthony's stain method, respectively. Our data allow us to conclude that the essential oil of P. amboinicus can be a good candidate for future research. PMID:22776104

In Brazil, since 2009, there has been an ever increasing widespread of the bla(KPC-2) gene, mainly in Klebsiellapneumoniae. This study aims to assess the molecular epidemiology and genetic background of this gene in Enterobacteriaceae (non-K. pneumoniae) species from 9 Brazilian states between 2009 and 2011. Three hundred eighty-seven isolates were analyzed exhibiting nonsusceptibility to carbapenems, in which the bla(KPC-2) gene was detected in 21.4%. By disk diffusion and E-test, these isolates exhibited high rates of resistance to most of the antimicrobials tested, including tigecycline (45.6% nonsusceptible) and polymyxin B (16.5%), the most resistant species being Enterobacter aerogenes and Enterobacter cloacae. We found great clonal diversity and a variety of bla(KPC-2)-carrying plasmids, all of them exhibiting a partial Tn4401 structure. Therefore, this study demonstrates the dissemination of KPC-2 in 9 Enterobacteriaceae species, including species that were not previously described such as Pantoea agglomerans and Providencia stuartii. PMID:25935630

The glnD gene in enteric bacteria encodes a uridylyltransferase/uridylyl-removing enzyme which acts as the primary nitrogen sensor in the nitrogen regulation (Ntr) system. We have investigated the role of this enzyme in transcriptional regulation of nitrogen fixation genes in Klebsiellapneumoniae by cloning glnD from this organism and constructing a null mutant by insertional inactivation of the chromosomal gene using the omega interposon. K. pneumoniae glnD encodes a 102.3 kDa polypeptide which is highly homologous to the predicted products of both Escherichia coli glnD and Azotobacter vinelandii nfrX. The glnD-omega mutant was unable to uridylylate PII and was altered in adenylylation/deadenylylation of glutamine synthetase. Uridylyltransferase was required for derepression of ntr-regulated promoters such as glnAp2 and pnifL but was not involved in the nif-specific response to changes in nitrogen status mediated by the nifL product. We conclude that a separate, as yet uncharacterised, nitrogen control system may be responsible for nitrogen sensing by NifL. PMID:7753028

The unique components of colostrum like free oligosaccharides and glycoconjugates are known to offer resistance to enzymatic digestion in the gastrointestinal tract and have the ability to inhibit the localized adherence of enteropathogens to the digestive tract of the neonates. In this context, we have evaluated the in vitro effect of buffalo colostrum immunoglobulin G on human pathogen Klebsiellapneumoniae, a predominant multidrug resistant pathogen associated with nasocomial infections. The investigation revealed growth inhibitory potential of immunoglobulin G in a dose dependent manner supported by scanning electron microscopic studies. The N-glycan enriched fraction of immunoglobulin G after PNGase treatment was found more effective, comparable to ampicillin than native immunoglobulin G supporting the fact that colostrum derived oligosaccharides is crucial and act as ideal substrates for undesirable and pathogenic bacteria. The MALDI TOF/TOF analysis confirmed the glycostructures of abundant N-glycans of immunoglobulin G exerting antibacterial activity. The proteomic analysis revealed variations between control and treated cells and expression of chemotaxis-CheY protein (14kDa) was evidenced in response to immunoglobulin G treatment. Hence, it would be interesting to investigate the mode of inhibition of multidrug-resistant K. pneumoniae by buffalo colostrum immunoglobulin G with the identification of a newly expressed signalling protein. PMID:27017977

The aims were to describe the genetic characterization of blaCTX-M-1 group gene in Klebsiellapneumoniae and to investigate the relationship between isolates by MLVA and PFGE. We analyzed 36 CTX-M group 1-ESBL producing K. pneumoniae. rmpA and wcaG virulence genes were identified by PCR. The genetic environment of blaCTX-M-1 was analyzed by PCR and sequencing. Plasmid replicons were determined using PCR-based replicon typing. The isolates were typed by MLVA and PFGE. All blaCTX-M-1 were blaCTX-M-15. The wcaG and rmpA were detected in 1 and 2 isolates, respectively. IncF were the most frequently detected replicons (63.88%). In all isolates, ISEcp1 was found upstream and orf477 downstream of blaCTX-M-15, IS26 was found in two isolates. MLVA identified 20 MLVA types, whereas PFGE identified 25 different profiles. The dissemination of CTX-M-15 in our isolates was due to the clonal spread of isolates and to the genetic transfer of mobile elements among unrelated strains. PMID:25734924

Glycerol bioconversion to 1,3-propanediol (1,3-PD) by Klebsiellapneumoniae (K. pneumoniae) can be characterized by an intricate network of interactions among biochemical fluxes, metabolic compounds, key enzymes and genetic regulatory. To date, there still exist some uncertain factors in this complex network because of the limitation in bio-techniques, especially in measuring techniques for intracellular substances. In this paper, among these uncertain factors, we aim to infer the transport mechanisms of glycerol and 1,3-PD across the cell membrane, which have received intensive interest in recent years. On the basis of different inferences of the transport mechanisms, we reconstruct various metabolic networks correspondingly and subsequently develop their dynamical systems (S-systems). To determine the most reasonable metabolic network from all possible ones, we establish a quantitative definition of biological robustness and undertake parameter identification and robustness analysis for each system. Numerical results show that it is most possible that both glycerol and 1,3-PD pass the cell membrane by active transport and passive diffusion.

A number of studies point to the conclusion that enterochelin, the iron chelator produced by a number of pathogenic enterobacteria, may be an essential metabolite for bacterial multiplication within the host. The compound removes iron from complexes with the host iron-binding proteins transferrin and lactoferrin, and the resulting ferric enterochelin is assimilated by the bacterial cell. It was reasoned that complexes of enterochelin with ions other than Fe3+ might act as antimetabolites and inhibit bacterial multiplication by interfering with the assimilation of ferric enterochelin. Enterochelin forms complexes with a number of group III and transition metal ions. The complex containing scandium exerts a bacteriostatic effect on Klebsiellapneumoniae in serum, whereas the indium complex induces a large increase in the generation time. The Fe3+ complexes of other microbial iron-transporting compounds are capable of reversing the bacteriostatic effect of the Sc3+ complex of enterochelin, suggesting that the compound acts solely by interfering with the enterochelin system of iron transport. Preliminary experiments show that the Sc3+ complex probably acts as a competitive inhibitor of ferric enterochelin. The Sc3+ complex of enterochelin exerts a therapeutic effect on intraperitoneal K. pneumoniae infections in mice similar to that obtained with kanamycin sulfate. PMID:6448022

In October 2012, an outbreak of gentamicin-resistant, ciprofloxacin non-susceptible extended-spectrum β-lactamase (ESBL)-producing Klebsiellapneumoniae occurred in a neonatal intensive care unit in Ireland. In order to determine whether the outbreak strain was more widely dispersed in the country, 137 isolates of K. pneumoniae with this resistance phenotype collected from 17 hospitals throughout Ireland between January 2011 and July 2013 were examined. ESBL production was confirmed phenotypically and all isolates were screened for susceptibility to 19 antimicrobial agents and for the presence of genes encoding bla TEM, bla SHV, bla OXA, and bla CTX-M; 22 isolates were also screened for bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48 genes. All isolates harboured bla SHV and bla CTX-M and were resistant to ciprofloxacin, gentamicin, nalidixic acid, amoxicillin-clavulanate, and cefpodoxime; 15 were resistant to ertapenem, seven to meropenem and five isolates were confirmed as carbapenemase producers. Pulsed-field gel electrophoresis of all isolates identified 16 major clusters, with two clusters comprising 61% of the entire collection. Multilocus sequence typing of a subset of these isolates identified a novel type, ST1236, a single locus variant of ST48. Data suggest that two major clonal groups, ST1236/ST48 (CG43) and ST15/ST14 (CG15) have been circulating in Ireland since at least January 2011. PMID:26113052

Community-acquired pyogenic liver abscess caused by Klebsiellapneumoniae is an emerging infectious disease. We explored the capsular polysaccharide synthesis (cps) regions of three non-K1, non-K2 K. pneumoniae strains, A1142, A7754, and A1517, from Taiwanese patients experiencing pyogenic liver abscess. Two of the strains, A1142 and A7754, belonged to capsular serotype K57, while the third belonged to a new capsular serotype, different from the previously reported 77 serotypes. Deletion and complementation experiments suggested that a unique K57 gene, a homologue of wzy, was essential for K57 capsular synthesis and confirmed that this gene cluster was a genetic coding region for K57. Compared to K1 and K2 strains, the three strains were all serum sensitive, suggesting that host factors might also be involved in the three patients. PCR using primers from specific genes for K57 was more sensitive and specific than traditional serotyping. The remaining strain, A1517, did not react to the antisera from any of the 77 serotypes, and none of the 77 reference strains reacted to the serum against this strain. Moreover, PCR analyses using various primer pairs from the serotype-specific open reading frames did not reveal cross-reactivity to any of the 77 reference strains, suggesting that this strain likely represents a new capsular type. We conclude that sequences from these two cps regions are very useful in detecting K57 and the new cps genotype. PMID:18508935

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiellapneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance. PMID:6351742

The microbial production of ethanol from D-xylose by a new soil isolate of Klebsiellapneumoniae and the mutants K. pneumoniae MB-16 and MB-16-1048 was studied. Kinetic and physiological properties of the mutants were compared with those of the original isolate. The volumetric rates of ethanol formation by mutants MB-16-1048 and MB-16 and the original isolate were 1.58, 0.50, and 0.06 g liter-1 h-1, respectively. The cultivation times of mutants MB-16-1048 and MB-16 were 20 and 18 h, respectively, and that of the original isolate was 118 h. Both the mutants exhibited metabolic similarities with the original isolate. Ethanol was the major end product of fermentation in all three strains. Acetic acid and carbon dioxide were the other two important by-products of fermentation. Pyruvic acid was accumulated in significant proportions as an intermediate. The proportion of pyruvate in the original isolate was 54% of the total D-xylose utilized, whereas for MB-16 and MB-16-1048 the values were about 42 and 22%, respectively. The lower fractions of pyruvate in mutants MB-16 and MB-16-1048 showed up as a 41 and 82% improvement, respectively, over the original isolate in terms of the ethanol yield. PMID:2502944

Riboswitches are cis-regulatory RNA elements on the mRNA level that control the expression of the downstream coding region. The interaction of the riboswitch with its specific metabolite, which is related to the function of the controlled gene, induces a structural change of the RNA architecture. Consequently, gene regulation is induced by un/masking of the ribosome binding site (RBS). In the genome of Klebsiellapneumoniae a sequence was identified by bioinformatics and proposed to be a B12 riboswitch regulated by coenzyme B12. Here we study this new coenzyme B12-dependent riboswitch system by in-line probing and ITC. The riboswitch sequence includes the whole expression platform as well as RBS. In-line probing experiments were performed to investigate the structural rearrangement of this 243-nt long RNA sequence while Isothermal Titration Calorimetry (ITC) yielded the thermodynamic parameters of the interaction between the riboswitch and its metabolite. The interaction of coenzyme B12 with the butB riboswitch of K. pneumoniae is an exothermic process with a 1:1 binding stoichiometry and binding affinities of log KA=6.73±0.02 at 15°C and log KA=6.00±0.09 at 30°C. PMID:26765998

Molecular features of the first carbapenem-resistant Klebsiellapneumoniae isolates (KP1 and KP2) from the University Hospital Tahar Sfar, Tunisia, were investigated. Antimicrobial susceptibility testing, multilocus sequence typing, S1 nuclease pulsed-field gel electrophoresis, Southern blot, and polymerase chain reaction (PCR)-based replicon typing were performed. Extended-spectrum β-lactamases and carbapenemase genes were detected by PCR and sequencing. Both isolates were multidrug resistant. KP1 was of sequence type (ST) ST101 and exhibited blaCTX-M-15 and blaTEM-1 on an untypeable plasmid and blaOXA-48 on an IncL/M plasmid. KP2 was genetically unrelated to KP1 (ST147) and harbored an IncA/C plasmid carrying blaCMY-4 and the blaOXA-48 derivative gene: blaOXA-204. This study reports the second case worldwide of an OXA-204-producing K. pneumoniae isolate from the same country, however, in a different genetic background. PMID:26447939

The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiellapneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K{sub i} of 30 {+-} 2 {micro}m. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.

The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues. The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiellapneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

The bioconverison of cellulose and hemicellulose substrates to 2,3-butanediol by a sequential coculture approach was investigated with the cellulolytic fungus Trichoderma harzianum E58 and the fermentative bacterium Klebsiellapneumoniae. Vogel medium optimal for the production of the cellulolytic and xylanolytic enzymes of the fungus was found to be inhibitory to butanediol fermentation. This inhibition appeared to be due to a synergistic effect of various ingredients, particularly the salts, present in the fungal medium. The removal or replacement of such ingredients from Vogel medium led to the relief of fermentation inhibition, but the treatments also resulted in a significant decrease in fungal enzyme production. Resting cells of K. pneumoniae could be used for butanediol production in the fungal medium, indicating that the inhibitory effect on solvent production under such conditions was due to the indirect result of growing inhibition of the bacterial cells. The resting-cell approach could be combined with a fed-batch system for the direct conversion of 8 to 10% (wt/vol) of Solka-Floc or aspenwood xylan to butanediol at over 30% of the theoretical conversion efficiencies.

Objective: There are few therapeutic options for treatment of multidrug resistant Klebsiellapneumoniae isolates as a hospital infectious agent (nosocomial infection). The aim of this study was to evaluate the antibacterial activity of Zataria multiﬂora Boiss extracts against ESBL-producing Klebsiellapneumoniae strains. Materials and Methods: This study was conducted on 100 K. pneumoniae isolates from two hospitals in Tehran, Iran. Antibiotic susceptibility tests were performed by Kirby-Bauer disc diffusion and microdilution broth methods and detection of ESBL was carried out according to CLSI guidelines. The blaCTX-M-15 plasmid gene was detected by PCR and sequencing methods. Extracts susceptibility test was performed by broth microdilution method. Results: Among 100 K. pneumoniae strains, 48 (48%) were ESBL positive. In this study, fosfomycin, colistin and tigecycline were more active than other antibiotics. The existence of blaCTX-M-15 was detected in 30 (62.5%) of 48 ESBL-producing isolates. The chloroformic extract showed potent activity against ESBL-producing K. pneumoniae strains (MIC50 = 1.56 mg/ml and MIC90=3.12mg/ml). The MIC50 and MIC90 (The MIC50 represents the MIC value at which ≥50% of the isolates in a test population are inhibited and the MIC90 represents the MIC value at which ≥90% of the strains within a test population are inhibited) were 3.12 and 6.25 mg/ml and 6.25 and 12.5 mg/ml for methanolic and acetonic extracts, respectively. Conclusion: The incidence of ESBL-producing K. pneumoniae is very high. Therefore, detection of ESBL-producing K. pneumoniae isolates is of great importance in identifying drug resistance patterns in K. pneumoniae isolates and in control of infections. Zataria multiflora may have the potential to be used against multidrug resistant organisms such as clinical isolates of ESBL-producing K. pneumoniae. PMID:27462557

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout (Il22Ra1Hep−/−) and Stat3 knockout (Stat3Hep−/−) mice were generated and subjected to intra-abdominal infection with Klebsiellapneumoniae, which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3. Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae, which was dependent on lipocalin 2 (LCN2). However, in vivo, rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti-K. pneumoniae bactericidal activity in vitro. These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections. PMID:26729763

Klebsiellapneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD50<100 CFU) while MGH 78578 is relatively avirulent. PMID:25203254

In spite of availability of moderately protective vaccine and antibiotics, new antibacterial agents are urgently needed to decrease the global incidence of Klebsiellapneumonia infections. MurF ligase, a key enzyme, which participates in the bacterial cell wall assembly, is indispensable to existence of K. pneumonia. MurF ligase lack mammalian vis-à-vis and have high specificity, uniqueness, and occurrence only in eubacteria, epitomizing them as promising therapeutic targets for intervention. In this study, we present a unified approach involving homology modeling and molecular docking studies on MurF ligase enzyme. As part of this study, a homology model of K. pneumonia (MurF ligase) enzyme was predicted for the first time in order to carry out structurebased drug design. The accuracy of the model was further validated using different computational approaches. The comparative molecular docking study on this enzyme was undertaken using different phyto-ligands from Desmodium sp. and a known antibiotic Ciprofloxacin. The docking analysis indicated the importance of hotspots (HIS 281 and ASN 282) within the MurF binding pocket. The Lipinski's rule of five was analyzed for all ligands considered for this study by calculating the ADME/Tox, drug likeliness using Qikprop simulation. Only ten ligands were found to comply with the Lipinski rule of five. Based on the molecular docking results and Lipinki values 6-Methyltetrapterol A was confirmed as a promising lead compound. The present study should therefore play a guiding role in the experimental design and development of 6-Methyltetrapterol A as a bactericidal agent. PMID:22715301

ABSTRACT Carbapenem-resistant Klebsiellapneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli. Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae. Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms. PMID:27353756

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout (Il22Ra1(Hep-/-)) and Stat3 knockout (Stat3(Hep-/-)) mice were generated and subjected to intra-abdominal infection with Klebsiellapneumoniae, which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3. Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae, which was dependent on lipocalin 2 (LCN2). However, in vivo, rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti-K. pneumoniae bactericidal activity in vitro. These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections. PMID:26729763

In spite of availability of moderately protective vaccine and antibiotics, new antibacterial agents are urgently needed to decrease the global incidence of Klebsiellapneumonia infections. MurF ligase, a key enzyme, which participates in the bacterial cell wall assembly, is indispensable to existence of K. pneumonia. MurF ligase lack mammalian vis-à-vis and have high specificity, uniqueness, and occurrence only in eubacteria, epitomizing them as promising therapeutic targets for intervention. In this study, we present a unified approach involving homology modeling and molecular docking studies on MurF ligase enzyme. As part of this study, a homology model of K. pneumonia (MurF ligase) enzyme was predicted for the first time in order to carry out structurebased drug design. The accuracy of the model was further validated using different computational approaches. The comparative molecular docking study on this enzyme was undertaken using different phyto-ligands from Desmodium sp. and a known antibiotic Ciprofloxacin. The docking analysis indicated the importance of hotspots (HIS 281 and ASN 282) within the MurF binding pocket. The Lipinski's rule of five was analyzed for all ligands considered for this study by calculating the ADME/Tox, drug likeliness using Qikprop simulation. Only ten ligands were found to comply with the Lipinski rule of five. Based on the molecular docking results and Lipinki values 6-Methyltetrapterol A was confirmed as a promising lead compound. The present study should therefore play a guiding role in the experimental design and development of 6-Methyltetrapterol A as a bactericidal agent. PMID:22715301

This study aimed to assess the frequency of Klebsiellapneumoniae in food samples and to detect antibiotic resistance phenotypes, antimicrobial resistance genes and the molecular subtypes of the recovered isolates. A total of 998 food samples were collected, and 99 (9.9%) K. pneumoniae strains were isolated; the frequencies were 8.2% (4/49) in fresh raw seafood, 13.8% (26/188) in fresh raw chicken, 11.4% (34/297) in frozen raw food and 7.5% (35/464) in cooked food samples. Antimicrobial resistance was observed against 16 antimicrobials. The highest resistance rate was observed for ampicillin (92.3%), followed by tetracycline (31.3%), trimethoprim-sulfamethoxazole (18.2%), and chloramphenicol (10.1%). Two K. pneumoniae strains were identified as extended-spectrum β-lactamase (ESBL)–one strain had three beta-lactamases genes (blaSHV, blaCTX-M-1, and blaCTX-M-10) and one had only the blaSHV gene. Nineteen multidrug-resistant (MDR) strains were detected; the percentage of MDR strains in fresh raw chicken samples was significantly higher than in other sample types (P<0.05). Six of the 18 trimethoprim-sulfamethoxazole-resistant strains carried the folate pathway inhibitor gene (dhfr). Four isolates were screened by PCR for quinolone resistance genes; aac(6’)-Ib-cr, qnrB, qnrA and qnrS were detected. In addition, gyrA gene mutations such as T247A (Ser83Ile), C248T (Ser83Phe), and A260C (Asp87Ala) and a parC C240T (Ser80Ile) mutation were identified. Five isolates were screened for aminoglycosides resistance genes; aacA4, aacC2, and aadA1 were detected. Pulsed-field gel electrophoresis-based subtyping identified 91 different patterns. Our results indicate that food, especially fresh raw chicken, is a reservoir of antimicrobial-resistant K. pneumoniae, and the potential health risks posed by such strains should not be underestimated. Our results demonstrated high prevalence, antibiotic resistance rate and genetic diversity of K. pneumoniae in food in China. Improved

This study aimed to assess the frequency of Klebsiellapneumoniae in food samples and to detect antibiotic resistance phenotypes, antimicrobial resistance genes and the molecular subtypes of the recovered isolates. A total of 998 food samples were collected, and 99 (9.9%) K. pneumoniae strains were isolated; the frequencies were 8.2% (4/49) in fresh raw seafood, 13.8% (26/188) in fresh raw chicken, 11.4% (34/297) in frozen raw food and 7.5% (35/464) in cooked food samples. Antimicrobial resistance was observed against 16 antimicrobials. The highest resistance rate was observed for ampicillin (92.3%), followed by tetracycline (31.3%), trimethoprim-sulfamethoxazole (18.2%), and chloramphenicol (10.1%). Two K. pneumoniae strains were identified as extended-spectrum β-lactamase (ESBL)-one strain had three beta-lactamases genes (blaSHV, blaCTX-M-1, and blaCTX-M-10) and one had only the blaSHV gene. Nineteen multidrug-resistant (MDR) strains were detected; the percentage of MDR strains in fresh raw chicken samples was significantly higher than in other sample types (P<0.05). Six of the 18 trimethoprim-sulfamethoxazole-resistant strains carried the folate pathway inhibitor gene (dhfr). Four isolates were screened by PCR for quinolone resistance genes; aac(6')-Ib-cr, qnrB, qnrA and qnrS were detected. In addition, gyrA gene mutations such as T247A (Ser83Ile), C248T (Ser83Phe), and A260C (Asp87Ala) and a parC C240T (Ser80Ile) mutation were identified. Five isolates were screened for aminoglycosides resistance genes; aacA4, aacC2, and aadA1 were detected. Pulsed-field gel electrophoresis-based subtyping identified 91 different patterns. Our results indicate that food, especially fresh raw chicken, is a reservoir of antimicrobial-resistant K. pneumoniae, and the potential health risks posed by such strains should not be underestimated. Our results demonstrated high prevalence, antibiotic resistance rate and genetic diversity of K. pneumoniae in food in China. Improved

Carbapenem resistance in Gram-negative bacteria is on the rise in the United States. A regional network was established to study microbiological and genetic determinants of clinical outcomes in hospitalized patients with carbapenem-resistant (CR) Klebsiellapneumoniae in a prospective, multicenter, observational study. To this end, predefined clinical characteristics and outcomes were recorded and K. pneumoniae isolates were analyzed for strain typing and resistance mechanism determination. In a 14-month period, 251 patients were included. While most of the patients were admitted from long-term care settings, 28% of them were admitted from home. Hospitalizations were prolonged and complicated. Nonsusceptibility to colistin and tigecycline occurred in isolates from 7 and 45% of the patients, respectively. Most of the CR K. pneumoniae isolates belonged to repetitive extragenic palindromic PCR (rep-PCR) types A and B (both sequence type 258) and carried either blaKPC-2 (48%) or blaKPC-3 (51%). One isolate tested positive for blaNDM-1, a sentinel discovery in this region. Important differences between strain types were noted; rep-PCR type B strains were associated with blaKPC-3 (odds ratio [OR], 294; 95% confidence interval [CI], 58 to 2,552; P < 0.001), gentamicin nonsusceptibility (OR, 24; 95% CI, 8.39 to 79.38; P < 0.001), amikacin susceptibility (OR, 11.0; 95% CI, 3.21 to 42.42; P < 0.001), tigecycline nonsusceptibility (OR, 5.34; 95% CI, 1.30 to 36.41; P = 0.018), a shorter length of stay (OR, 0.98; 95% CI, 0.95 to 1.00; P = 0.043), and admission from a skilled-nursing facility (OR, 3.09; 95% CI, 1.26 to 8.08; P = 0.013). Our analysis shows that (i) CR K. pneumoniae is seen primarily in the elderly long-term care population and that (ii) regional monitoring of CR K. pneumoniae reveals insights into molecular characteristics. This work highlights the crucial role of ongoing surveillance of carbapenem resistance determinants. PMID:24798270

We present a rare case of invasive liver abscess syndrome due to Klebsiellapneumoniae (K. pneumoniae) with metastatic meningitis and septic shock. A previously healthy, 55-year-old female patient developed fever, liver abscess, septic shock, purulent meningitis and metastatic hydrocephalus. Upon admission, the clinical manifestations, laboratory and imaging examinations were compatible with a diagnosis of K. pneumoniae primary liver abscess. Her distal metastasis infection involved meningitis and hydrocephalus, which could flare abruptly and be life threatening. Even with early adequate drainage and antibiotic therapy, the patient’s condition deteriorated and she ultimately died. To the best of our knowledge, this is the first case of K. pneumoniae invasive liver abscess syndrome with septic meningitis reported in mainland China. Our findings reflect the need for a better understanding of the epidemiology, risk factors, complications, comorbid medical conditions and treatment of this disease. PMID:26973425

We present a rare case of invasive liver abscess syndrome due to Klebsiellapneumoniae (K. pneumoniae) with metastatic meningitis and septic shock. A previously healthy, 55-year-old female patient developed fever, liver abscess, septic shock, purulent meningitis and metastatic hydrocephalus. Upon admission, the clinical manifestations, laboratory and imaging examinations were compatible with a diagnosis of K. pneumoniae primary liver abscess. Her distal metastasis infection involved meningitis and hydrocephalus, which could flare abruptly and be life threatening. Even with early adequate drainage and antibiotic therapy, the patient's condition deteriorated and she ultimately died. To the best of our knowledge, this is the first case of K. pneumoniae invasive liver abscess syndrome with septic meningitis reported in mainland China. Our findings reflect the need for a better understanding of the epidemiology, risk factors, complications, comorbid medical conditions and treatment of this disease. PMID:26973425

This study evaluated the efficacy of tigecycline (TIG), polymyxin B (PMB), and meropenem (MER) in 80 rats challenged with Klebsiellapneumoniae carbapenemase (KPC)-producing K. pneumoniae infection. A time-kill assay was performed with the same strain. Triple therapy and PMB+TIG were synergistic, promoted 100% survival, and produced negative peritoneal cultures, while MER+TIG showed lower survival and higher culture positivity than other regimens (P = 0.018) and was antagonistic. In vivo and in vitro studies showed that combined regimens, except MER+TIG, were more effective than monotherapies for this KPC-producing strain. PMID:25896686

Klebsiellapneumoniae J1 is a Gram-negative strain, which belongs to a protein-based microbial flocculant-producing bacterium. However, little genetic information is known about this species. Here we carried out a whole-genome sequence analysis of this strain and report the complete genome sequence of this organism and its genetic basis for carbohydrate metabolism, capsule biosynthesis and transport system. PMID:26806487

Antibiotic resistance in Klebsiellapneumoniae strains is an increasing problem in a lot of hospitals. It is a public health emergency because it relates with high mortality rate among patients in Intensive Care Unit (ICU). From 1/1/2009 to 31/08/2010, in ICU of SS Annunziata Hospital of Taranto, 140 isolated Klebsiellapneumoniae strains were detected. The strain identification and antimicrobial susceptibility testing were performed using a Vitek2 automated system. These isolate showed a low level of susceptibility to levofloxacin (3.4%), ciprofloxacin (6.2%), ceftazidime (2.8%) and piperacillin/tazobactam (8%). We reported also that the 10% and 13.9% of them were susceptible to meropenem and imipenem. An anti-Klebsiellapneumoniae activity in vitro to tigecycline was present in 64.6% of isolates while almost all strains (56/58) tested to colistin were susceptible. In order to our data of worryng high multiclass drug resistance including tygecicline, it needs to apply appropriate measures of surveillance and antibiotic prescription to avoid rapid spread of these mutiresistant strains in other areas. PMID:22825382

Background Nosocomial infections caused by Carbapenem-resistant Klebsiellapneumoniae (CRKP) are increasing. Our aim in this study was to investigate the risk factors of CRKP infections. Material/Methods A retrospective cohort study was performed between 1 January and 31 December 2012 in ICU patients. Data was taken from the hospital infection control database for CRKP. The clinical samples collected from the patients were tested by an automatized system and disk diffusion. SPSS software v11.5 was used for statistical analysis. Results Totally, 105 Klebsiellapneumoniae isolates were found in 2012 and the carbapenem resistance rate was 48%. The first episode of infection was taken into risk factor analysis. Of the 98 patients, 61 (62.2%) were male and the mean and median ages were 30.4±29.8 and 25 (0–93). The length of stay was longer in the resistant group (p=0.026). Mortality was 48% in the whole group and similar between groups (p=0.533). There was a relationship between meropenem and third-generation cephalosporin use and resistance (OR 3.244 (1.193–8.819) and OR: 3.590 (1.056–12.209). The other risk factors in univariate analysis were: Immunosuppression OR: 2.186 (1.754–2.724), nasogastric catheter OR: 3.562 (1.317–9.634), peripheral arterial catheter OR: 2.545 (1.027–6.307), and being admitted to the neurosurgical unit OR: 4.324 (1.110–16.842). The multivariate analysis showed use of third-generation cephalosporin OR: 4.699 (1.292–17.089), nasogastric catheter use OR: 3.983 (1.356–11.698), and being admitted to neurosurgical ICU OR: 4.603 (1.084–19.555) as independent risk factors. Conclusions Restriction of third-generation cephalosporin and carbapenem use and invasive procedures, along with infection control precautions and disinfection policies, may be effective in reducing the carbapenem resistance in ICUs. PMID:25595166

Klebsiellapneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiellapneumoniae Carbapenemase (KPC)-encoding gene blaKPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain blaKPC-2 and blaNDM-1genes simultaneously in the isolate. Our data showed the high prevalence of blaKPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis

The aim of this study was to identify factors associated with mortality in intensive care unit patients with Klebsiellapneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) septic shock. A retrospective analysis of intensive care unit patients with KPC-Kp infection and septic shock observed in a large teaching hospital from November 2010 to December 2014 was performed. A total of 111 patients were included in the study. The most frequent source of infection was unknown-focus bacteraemia in 53 patients (47.7%). The rate of resistance to colistin was 51.3%; 30-day mortality was reported for 44 patients (39.6%). Surviving patients were more frequently treated with an initial therapy (within 24 hours) including two or more antibiotics displaying in vitro activity against the isolated KPC-Kp strain (41.8 vs. 18.1%, p 0.01) and were also more likely to receive a definitive therapy including two or more in vitro active antibiotics (85.1 vs. 15.9%, p <0.001). Cox regression analysis revealed that a colistin-containing antibiotic regimen (hazard ratio (HR) 0.21, confidence interval (CI) 95% 0.05-0.72, p <0.001), use of two or more in vitro active antibiotics as definite therapy (HR 0.08, CI 95% 0.02-0.21, p <0.001) and control of removable source of infection (HR 0.14, CI 95% 0.04-0.25, p <0.001) were associated with favourable outcome; colistin resistance (HR 8.09, CI 95% 3.14-11.23, p 0.001) and intra-abdominal source of infection (HR 2.92, CI 95% 2.11-4.12, p 0.002) were associated with death. In conclusion, use of a definitive therapy with at least two antibiotics displaying in vitro activity against the KPC-Kp isolates was the most important determinant of favourable outcome, whilst isolation of colistin-resistant strains was associated with death in septic patients with KPC-Kp infection. PMID:26850826

We describe a large hospital outbreak (93 bloodstream infections) of colistin-resistant Klebsiellapneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates which was mirrored by increased colistin consumption. The outbreak was mostly traced to the clonal expansion of an mgrB deletion mutant of an ST512 strain that produced KPC-3. PMID:26202124

We report the first case of a child in the United States infected with an organism producing a Verona Integron-Encoded Metallo-β-Lactamase. This child succumbed to a ventilator-associated pneumonia caused by a Klebsiellapneumoniae producing this resistance mechanism. PMID:27147714

Previous studies reported decreased mortality in patients with carbapenemase-producing Klebsiellapneumoniae bloodstream infections (BSIs) treated with combination therapy but included carbapenem-susceptible and -intermediate isolates, as per revised CLSI breakpoints. Here, we assessed outcomes in patients with BSIs caused by phenotypically carbapenem-resistant K. pneumoniae (CRKP) according to the number of in vitro active agents received and whether an extended-spectrum beta-lactam (BL) antibiotic, including meropenem, or an extended-spectrum cephalosporin was administered. We retrospectively reviewed CRKP BSIs at two New York City hospitals from 2006 to 2013, where all isolates had meropenem or imipenem MICs of ≥4 μg/ml. Univariate and multivariable models were created to identify factors associated with mortality. Of 141 CRKP BSI episodes, 23% were treated with a single active agent (SAA), 26% were treated with an SAA plus BL, 28% were treated with multiple active agents (MAA), and 23% were treated with MAA plus BL. Ninety percent of isolates had meropenem MICs of ≥16 μg/ml. Thirty-day mortality was 33% overall and did not significantly differ across the four treatment groups in a multivariable model (P = 0.4); mortality was significantly associated with a Pitt bacteremia score of ≥4 (odds ratio [OR], 7.7; 95% confidence interval [CI], 3.2 to 18.1; P = 0.1), and immunosuppression was protective (OR, 0.4; 95% CI, 0.2 to 1.0; P = 0.04). Individual treatment characteristics were also not significantly associated with outcome, including use of SAAs versus MAA (26% versus 38%, P = 0.1) or BL versus no BL (26% versus 39%, P = 0.1). In summary, in patients with CRKP BSIs caused by isolates with high carbapenem MICs, the role of combination therapy remains unclear, highlighting the need for prospective studies to identify optimal treatment regimens. PMID:27044555

Klebsiellapneumoniae KCTC2242 has high potential in the production of a high-value chemical, 2,3-butanediol (2,3-BDO). However, accumulation of metabolites such as lactate during cell growth prevent large-scale production of 2,3-BDO. Consequently, we engineered K. pneumoniae to redistribute its carbon flux toward 2,3-BDO production. The ldhA gene deletion and gene overexpression (budA and budB) were conducted to block a pathway that competitively consumes reduced nicotinamide adenine dinucleotide and to redirect carbon flux toward 2,3-BDO biosynthesis, respectively. These steps allowed efficient glucose conversion to 2,3-BDO under slightly acidic conditions (pH 5.5). The engineered strain SGSB105 showed a 40% increase in 2,3-BDO production from glucose compared with that of the host strain, SGSB100. Genes closely related to 2,3-BDO biosynthesis were observed at the gene transcription level by cultivating the SGSB100, SGSB103, SGSB104, and SGSB105 strains under identical growth conditions. Transcription levels for budA, budB, and budC increased approximately 10% during the log phase of cell growth relative to that of SGSB100. Transcription levels of 2,3-BDO genes in SGSB105 remained high during the log and stationary phases. Thus, the carbon flux was redirected toward 2,3-BDO production. Data on batch culture and gene transcription provide insight into improving the metabolic network for 2,3-BDO biosynthesis for industrial applications. PMID:25329548

The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiellapneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks. PMID:8890231

Biogenesis of pyrroloquinoline quinone (PQQ) in Klebsiellapneumoniae requires the expression of six genes (pqqA-F). One of these genes (pqqE) encodes a 43 kDa protein (PqqE) that plays a role in the initial steps in PQQ formation (Veletrop et al. (1995) J. Bacteriol. 177, 5088-5098). PqqE contains two highly conserved cysteine motifs at the N and C-termini, with the N-terminal motif comprised of a consensus sequence of CX3CX2C that is unique to a family of proteins known as radical S-adenosyl-L-methionine (SAM) enzymes (Sofia et al. (2001) Nucleic Acids Res. 29, 1097-1106). PqqE from K. pneumoniae was cloned into E. coli and expressed as the native protein and with an N-terminal His6-tag. Anaerobic expression and purification of the His6-tag PqqE results in an enzyme with a brownish-red hue indicative of Fe-S cluster formation. Spectroscopic and physical analyses indicate that PqqE contains a mixture of Fe-S clusters, with the predominant form of the enzyme containing two [4Fe-4S] clusters. PqqE isolated anaerobically yields active enzyme capable of cleaving SAM to methionine and 5′-deoxyadenosine in an uncoupled reaction (kobs = 0.011 ± 0.001 min-1). In this reaction, the 5′-deoxyadenosyl radical either abstracts a hydrogen atom from a solvent accessible position in the enzyme or obtains a proton and electron from buffer. The putative PQQ substrate PqqA has not yet been shown to be modified by PqqE, implying either that PqqA must be modified before becoming the substrate for PqqE and/or that another protein in the biosynthetic pathway is critical for the initial steps in PQQ biogenesis. PMID:19746930

The production of Klebsiellapneumoniae carbapenemases (KPCs) by Enterobacteriaceae has become a significant problem in recent years. To identify factors that could predict isolation of KPC-producing K. pneumoniae (KPCKP) in clinical samples from hospitalized patients, we conducted a retrospective, matched (1:2) case-control study in five large Italian hospitals. The case cohort consisted of adult inpatients whose hospital stay included at least one documented isolation of a KPCKP strain from a clinical specimen. For each case enrolled, we randomly selected two matched controls with no KPCKP-positive cultures of any type during their hospitalization. Matching involved hospital, ward, and month/year of admission, as well as time at risk for KPCKP isolation. A subgroup analysis was also carried out to identify risk factors specifically associated with true KPCKP infection. During the study period, KPCKP was isolated from clinical samples of 657 patients; 426 of these cases appeared to be true infections. Independent predictors of KPCKP isolation were recent admission to an intensive care unit (ICU), indwelling urinary catheter, central venous catheter (CVC), and/or surgical drain, ≥2 recent hospitalizations, hematological cancer, and recent fluoroquinolone and/or carbapenem therapy. A Charlson index of ≥3, indwelling CVC, recent surgery, neutropenia, ≥2 recent hospitalizations, and recent fluoroquinolone and/or carbapenem therapy were independent risk factors for KPCKP infection. Models developed to predict KPCKP isolation and KPCKP infection displayed good predictive power, with the areas under the receiver-operating characteristic curves of 0.82 (95% confidence interval [CI], 0.80 to 0.84) and 0.82 (95% CI, 0.80 to 0.85), respectively. This study provides novel information which might be useful for the clinical management of patients harboring KPCKP and for controlling the spread of this organism. PMID:24733460

The emergence of Klebsiellapneumoniae carbapenemases (KPCs) producing bacteria has become a significant global public health challenge while the optimal treatment remains undefined. We performed a systematic review of published studies and reports of treatment outcomes of KPC infections using MEDLINE (2001–2011). Articles or cases were excluded if one of the following was fulfilled: no individual patient data provided, no treatment regimen specified, no treatment outcome specified, report of colonization, or greater than three antibiotics were used to treat the KPC infection. Data extracted included patient demographics, site of infection, organism, KPC subtype, antimicrobial therapy directed at KPC-infection, and treatment outcome. Statistical analysis was performed in an exploratory manner. A total of 38 articles comprising 105 cases were included in the analysis. The majority of infections were due to K. pneumoniae (89%). The most common site of infection was blood (52%), followed by respiratory (30%), and urine (10%). Forty-nine (47%) cases received monotherapy and 56 (53%) cases received combination therapy directed at the KPC-infection. Significantly more treatment failures were seen in cases that received monotherapy compared to cases who received combination therapy (49% vs 25%; p= 0.01). Respiratory infections were associated with higher rates of treatment failure with monotherapy compared to combination therapy (67% vs 29% p= 0.03). Polymyxin monotherapy was associated with higher treatment failure rates compared to polymyxin-based combination therapy (73% vs 29%; p= 0.02); similarly, higher treatment failure rates were seen with carbapenem monotherapy compared to carbapenem-based combination therapy (60% vs 26%; p= 0.03). Overall treatment failure rates were not significantly different in the three most common antibiotic-class combinations: polymyxin plus carbapenem, polymyxin plus tigecycline, polymyxin plus aminoglycoside (30%, 29%, and 25

Bacterium Klebsiellapneumoniae (KP) contains a prominent capsule. Clinical infections usually are associated with pneumonia or urinary tract infection (UTI). Emerging evidence implicates KP in severe liver abscess especially in diabetic patients. The goal of this study was to investigate the capsular polysaccharides from KP of liver abscess (hepatic-KP) and of UTI-KP. The composition of capsular polysaccharides was analyzed by capillary high-performance liquid chromatography (HPLC, Dionex system). The terminal sugars were assayed by binding ability to lectins. The results showed that the capsule of a hepatic KP (KpL1) from a diabetic patient contained fucose, while the capsule from UTI-KP (KpU1) did not. The absence of fucose was verified by the absence of detectable polymerase chain reaction (PCR) fragment for fucose synthesis genes, gmd and wcaG in KpU1. Mice infected with the KpL1 showed high fatality, whereas those infected with the KpU1 showed high survival rate. The KpL1 capsule was reactive to lectins AAA and AAL, which detect fucose, while the KpU1 capsule was reactive to lectin GNA, which detects mannose. Phagocytosis experiment in mouse peritoneal cavity indicated that the peritoneal macrophages could interact with KpU1, while rare association of KpL1 with macrophages was observed. This study revealed that different polysaccharides were displayed on the bacterial capsules of virulent KpL1 as compared with the less virulent KpU1. Interaction of KpU1 with mice peritoneal macrophages was more prominent than that of KpL1. The possession of fucose might contribute to KpL1 virulence by avoiding phagocytosis since fucose on bacteria had been implicated in immune evasion. PMID:18156307

This study was conducted to investigate the prevalence and characteristics of ertapenem-resistant (ETP-R) Klebsiellapneumoniae isolates at a Taiwanese hospital. The disk-diffusion tests revealed that the rate of ertapenem resistance among all isolates collected in 2008 was 13.5%, and the resistance rate among bloodstream isolates increased from 0% to 13.6% between 2001 and 2008. Eighty-two nonduplicate ETP-R isolates collected in 2008 were examined. Seventy-four (90.2%) isolates of them had extended-spectrum β-lactamases (CTX-M- and SHV-type), AmpC enzymes (DHA-1 and CMY-2), and IMP-8 metallo-β-lactamase alone or in combination, and an extremely high prevalence of fluoroquinolone resistance (95.1%) and plasmid-mediated quinolone resistance determinants (90.2%) were also observed. Eighteen ETP-R but imipenem-susceptible isolates were selected and compared with 18 imipenem-nonsusceptible isolates collected before 2008. Sequence analyses revealed genetic disruptions of OmpK36 in 11 imipenem-nonsusceptible and 6 imipenem-susceptible isolates, respectively, and OmpK35 disruptions in 10 isolates for both groups. For the isolates with intact ompK36, sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests decreased expression of OmpK36 in 5 of 7 imipenem-nonsusceptible isolates and 3 of 12 imipenem-susceptible isolates. In conclusion, the increasing prevalence of ertapenem resistance that was predominantly attributed to noncarbapenemase-mediated resistance mechanisms in K. pneumoniae is becoming a serious treat to patients in Taiwan. PMID:21352075

The inhibition potentials of products and substrate on the growth of Clostridium butyricum and Klebsiellapneumoniae in the glycerol fermentation are examined from experimental data and with a mathematical model. Whereas the inhibition potential of externally added and self-produced 1,3-propanediol is essentially the same, butyric acid produced by the culture is more toxic than that externally added. The same seems to apply for acetic acid. The inhibitory effect of butyric acid is due to the total concentration instead of its undissociated form. For acetic acid, it cannot be distinguished between the total concentration and the undissociated form. The inhibition effects of products and substrate in the glycerol fermentation are irrespective of the strains, and, therefore, the same growth model can be used. The maximum product concentrations tolerated are 0.35 g/L for undissociated acetic acid, 10.1 g/L for total butyric acid, 16.6 g/L for ethanol, 71.4 g/L for 1,3-propanediol, and 187.6 g/L for glycerol, which are applicable to C. butyricum and K. pneumoniae growth under a variety of conditions. For 55 steady-states, which were obtained from different types of continuous cultures over a pH range of 5.3--8.5 and under both substrate limitation and substrate excess, the proposed growth model fits the experimental data with an average deviation of 17.0%. The deviation of model description from experimental values reduces of 11.4% if only the steady-states with excessive substrate are considered.

The effects of starvation on bacterial penetration through artificial rock cores were examined. Klebsiellapneumoniae was starved in a simple salts solution for a duration of up to 4 weeks. These cell suspensions were injected into sintered glass bead cores, and the resulting reductions in core permeabilities were recorded. Vegetative cell cultures of K. pneumoniae grown in a sodium citrate medium were injected into other, similar cores, and the reductions in core permeabilities were recorded. The starved cell suspensions did not completely block the core pores, whereas the vegetative cultures reduced core permeability to less than 1%. Scanning electron microscopy of core section infiltrated with either vegetative or starved cells showed that the former produced shallow skin plugs and copious amounts of glycocalyx at the inlet face, whereas the latter produced very little glycocalyx and the cells were distributed evenly throughout the length of the core. The use of a DNA assay to produce a cell distribution profile showed that, compared with the vegetative cells, starved bacteria were able to penetrate deeper into the cores. This was due to the smaller size of the cells and the reduction in biofilm production. This ability of starved bacteria to penetrate further into cores than the normal-size vegetative cells can be usefully applied to selective plugging for enhanced oil recovery. To further test the suitability of starved cells for use in selective plugging, the activities of starved cells present within cores were monitored before and after nutrient stimulation. Our data indicate that with nutrient stimulation, the starved cells lose their metabolic dormancy and produce reductions in core permeability due to cell growth and polymer production.

The dha regulon in Klebsiellapneumoniae enables the organism to grown anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydoxyacetone and was screened for the production of 1, 3-PD. The cosmid pTC1 (42.5 kn total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycersol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1, 3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.

The spread of carbapenem-non-susceptible Klebsiellapneumoniae strains bearing different resistance determinants is a rising problem worldwide. Especially infections with KPC (Klebsiellapneumoniae carbapenemase) - producers are associated with high mortality rates due to limited treatment options. Recent clinical studies of KPC-blood stream infections revealed that colistin-based combination therapy with a carbapenem and/or tigecycline was associated with significantly decreased mortality rates when compared to colistin monotherapy. However, it remains unclear if these observations can be transferred to K. pneumoniae harboring other mechanisms of carbapenem resistance. A three-dimensional synergy analysis was performed to evaluate the benefits of a triple combination with meropenem, tigecycline and colistin against 20 K. pneumoniae isolates harboring different β-lactamases. To examine the mechanism behind the clinically observed synergistic effect, efflux properties and outer membrane porin (Omp) genes (ompK35 and ompK36) were also analyzed. Synergism was found for colistin-based double combinations for strains exhibiting high minimal inhibition concentrations against all of the three antibiotics. Adding a third antibiotic did not result in further increased synergistic effect in these strains. Antagonism did not occur. These results support the idea that colistin-based double combinations might be sufficient and the most effective combination partner for colistin should be chosen according to its MIC. PMID:26067824

Klebsiellapneumoniae mastitis in dairy cattle is generally due to an opportunistic infection from the environment, resulting in large heterogeneity among mastitis-causing strains within a herd. However, in mastitis outbreaks in 4 herds, several strains of K. pneumoniae were identified as the cause of infection in multiple cows, suggesting increased ability to either cause disease or evade host defenses. In this study, differences in capsule formation and immune evasion were compared in 5 pairs of K. pneumoniae strains, where one strain in each pair was associated with multiple cases of mastitis and the other with a single case of mastitis. Production of capsular polysaccharide, ability to evade killing by polymorphonuclear neutrophilic leukocytes (PMNL), and the relationship between the 2 were evaluated for each strain grown in broth or milk. Growth of isolates in skim milk increased capsule size and ability to evade killing by PMNL, depending on strain type. Specifically, strains associated with multiple cases of mastitis had increased capsule size in skim milk. Strains associated with single cases of mastitis were better able to evade killing by PMNL when grown in skim milk. Our results, although preliminary, suggest that the 2 groups of strains may constitute different subpopulations of K. pneumoniae. However, our findings do not indicate that capsule or evasions of killing by PMNL explain increased mastitis outbreaks with Klebsiella. Further work will explain the enhanced ability of some strains to cause mastitis in dairy cows. PMID:24534505

2,3-Butanediol is one of the promising bulk chemicals with wide applications. Its fermentative production has attracted great interest due to the high end concentration. However, large-scale production of 2,3-butanediol requires low-cost substrate and efficient fermentation process. In the present study, 2,3-butanediol production by Klebsiellapneumoniae from Jerusalem artichoke tubers was successfully performed, and various technologies, including separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), were investigated. The concentration of target products reached 81.59 and 91.63 g/l, respectively after 40 h in batch and fed-batch SSF processes. Comparing with fed-batch SHF, the fed-batch SSF provided 30.3% higher concentration and 83.2% higher productivity of target products. The results showed that Jerusalem artichoke tuber is a favorable substrate for 2,3-butanediol production, and the application of fed-batch SSF for its conversion can result in a more cost-effective process. PMID:19122999

An increase in the antibiotic resistance among members of the Enterobacteriaceae family has been observed worldwide. Multidrug-resistant Gram-negative rods are increasingly reported. The treatment of infections caused by Escherichia coli and other Enterobacteriaceae has become an important clinical problem associated with reduced therapeutic possibilities. Antimicrobial carbapenems are considered the last line of defense against multidrug-resistant Gram-negative bacteria. Unfortunately, an increase of carbapenem resistance due to the production of Klebsiellapneumoniae carbapenemase (KPC) enzymes has been observed. In this study we describe the ability of E. coli to produce carbapenemase enzymes based on the results of the combination disc assay with boronic acid performed according to guidelines established by the European Community on Antimicrobial Susceptibility Testing (EUCAST) and the biochemical Carba NP test. Moreover, we evaluated the presence of genes responsible for the production of carbapenemases (blaKPC, blaVIM, blaIMP, blaOXA-48) and genes encoding other β-lactamases (blaSHV, blaTEM, blaCTX-M) among E. coli isolate. The tested isolate of E. coli that possessed the blaKPC-3 and blaTEM-34 genes was identified. The tested strain exhibited susceptibility to colistin (0.38 μg/mL) and tigecycline (1 μg/mL). This is the first detection of blaKPC-3 in an E. coli ST479 in Poland. PMID:26339599

In recent years, an emergent Klebsiellapneumoniae hypermucoviscosity (HMV) phenotype has been associated with increased invasiveness and pathogenicity in primates. In this project, bacteria recovered from infected African green monkeys (AGM) (Chlorocebus aethiops sabaeus) were screened for HMV phenotype, and were compared to non-HMV isolates in in vitro, serum, and oxidative-mediated killing assays. Complement-mediated killing was assessed utilizing freshly collected serum from healthy AGM. Oxidative-mediated killing was investigated utilizing sodium hypochlorite and hydrogen peroxide. Compared to non-HMV isolates, HMV isolates were more resistant to serum-mediated and oxidative killing (p<0.05). Phagocytosis resistance was evaluated using AGM peripheral blood monocytes (PBMC), and results indicated that non-HMV isolates associated with the AGM PBMC to a greater extent than HMV isolates (p<0.001). Measurement of lactate dehydrogenase release showed that HMV isolates were more cytotoxic to AGM PBMC than non-HMV isolates (p<0.001). Thus, the hypermucoid phenotype appears to be an important virulence factor that promotes evasion of innate immune defenses. PMID:25614101

Summary: The spread of Enterobacteriaceae, primarily Klebsiellapneumoniae, producing KPC, VIM, IMP, and NDM carbapenemases, is causing an unprecedented public health crisis. Carbapenemase-producing enterobacteria (CPE) infect mainly hospitalized patients but also have been spreading in long-term care facilities. Given their multidrug resistance, therapeutic options are limited and, as discussed here, should be reevaluated and optimized. Based on susceptibility data, colistin and tigecycline are commonly used to treat CPE infections. Nevertheless, a review of the literature revealed high failure rates in cases of monotherapy with these drugs, whilst monotherapy with either a carbapenem or an aminoglycoside appeared to be more effective. Combination therapies not including carbapenems were comparable to aminoglycoside and carbapenem monotherapies. Higher success rates have been achieved with carbapenem-containing combinations. Pharmacodynamic simulations and experimental infections indicate that modification of the current patterns of carbapenem use against CPE warrants further attention. Epidemiological data, though fragmentary in many countries, indicate CPE foci and transmission routes, to some extent, whilst also underlining the lack of international collaborative systems that could react promptly and effectively. Fortunately, there are sound studies showing successful containment of CPE by bundles of measures, among which the most important are active surveillance cultures, separation of carriers, and assignment of dedicated nursing staff. PMID:23034326

2-Ketogluconic acid production by Klebsiellapneumoniae is a pH-dependent process, strictly proceeding under acidic conditions. Unfortunately, cell growth is inhibited by acidic conditions, resulting in low productivity of 2-ketogluconic acid. To overcome this deficiency, a two-stage fermentation strategy was exploited in the current study. During the first stage, the culture was maintained at neutral pH, favoring cell growth. During the second stage, the culture pH was switched to acidic conditions favoring 2-ketogluconic acid accumulation. Culture parameters, including switching time, dissolved oxygen levels, pH, and temperature were optimized for the fed-batch fermentation. Characteristics of glucose dehydrogenase and gluconate dehydrogenase were revealed in vitro, and the optimal pHs of the two enzymes coincided with the optimum culture pH. Under optimum conditions, a total of 186 g/l 2- ketogluconic acid was produced at 26 h, and the conversion ratio was 0.98 mol/mol. This fermentation strategy has successfully overcome the mismatch between optimum parameters required for cell growth and 2-ketogluconic acid accumulation, and this result has the highest productivity and conversion ratio of 2-ketogluconic and produced by microorganism. PMID:24572278

An exopolysaccharide (KNPS) of an average molecular weight ∼1.8×10(5) Da was isolated from the culture medium of Klebsiellapneumoniae PB12. Structural characterization of KNPS was carried out using sugar and methylation analysis, Smith degradation and 1D/2D NMR experiments. Sugar analysis showed that the KNPS composed of arabinose, galactose, 3-O-methyl-galctose and glucose in a molar ratio of nearly 4:3:1:1. The proposed repeating unit of the KNPS has a backbone chain consisting of two (1→6)-galactopyranosyl residues, two (1→5)-arabinofuranosyl residues, one (1→6)-glucopyranosyl residue and one (1→3)-arabinopyranosyl residue, out of which one (1→6)-galactopyranosyl residue was branched at O-2 position with a (1→2)-linked-galactopyranosyl residue terminated with non reducing arabinofuranosyl residue and one (1→5)-arabinofuranosyl residue branched at O-3 position with non reducing end 3-O-Me-galactopyranosyl residue. KNPS was found non-toxic toward human lymphocyte up to the dosage of 100 μg/ml. KNPS enhanced malondialdehyde (MDA), reactive oxygen species (ROS), and have the potential to alter the ratio of oxidized glutathione (GSSG) and reduced glutathione (GSH) levels in the cellular system. PMID:25999015

A cosmid clone which was capable of depleting acetoin in vivo was isolated from a library of Klebsiellapneumoniae CG43 cosmids. The smallest functional subclone contained a 3.9-kb DNA fragment of the cosmid clone. Sequencing of the DNA fragment revealed three open reading frames (ORFs A, B, and C) encoding polypeptides of 34, 36, and 52 kDa, respectively. The presence of these proteins was demonstrated by expression of the recombinant DNA clone in Escherichia coli. Considerable similarities between the deduced amino acid sequences of the ORFs and those of the following enzymes were found: acetoin dissimilation enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, and branched-chain 2-oxo acid dehydrogenase complex of various origins. Activities of these enzymes, including acetoin-dependent dichlorophenolin-dohenol oxidoreductase and dihydrolipoamide acetyltransferase, were detected in the extracts of E. coli harboring the genes encoding products of the three ORFs. Although not required for acetoin depletion in vivo, a possible fourth ORF (ORF D), located 39 nucleotides downstream of ORF C, was also identified. The deduced N-terminal sequence of the ORF D product was highly homologous to the dihydrolipoamide dehydrogenases of several organisms. Primer extension analysis identified the transcriptional start of the operon as an A residue 72 nucleotides upstream of ORF A. Images PMID:8206829

The effects of both biomass age and cell recycling on the 1,3-propanediol (1,3-PDO) production by Klebsiellapneumoniae were investigated in a membrane-supported bioreactor using hollow-fiber ultrafiltration membrane module in two separate experiments. It was determined that older cells have a negative effect on 1,3-PDO production. The concentrations of by-products, such as acetic acid and ethanol, increased in cultures with older cells, whereas the concentrations of succinic acid, lactic acid and 2,3-butanediol decreased. The effect of cell recycling was comparatively studied at a cell recycling ratio of 100 %. The results showed that cell recycling had also negative effects on 1,3-PDO fermentation. It was hypothesized that both cell recycling and biomass age caused metabolic shifts to undesired by-products which then inhibited the 1,3-PDO production. On the other hand, the use of hollow-fiber ultrafiltration membrane module was found to be very effective in terms of removal of cells from the fermentation broth. PMID:23892658

An increase in the antibiotic resistance among members of the Enterobacteriaceae family has been observed worldwide. Multidrug-resistant Gram-negative rods are increasingly reported. The treatment of infections caused by Escherichia coli and other Enterobacteriaceae has become an important clinical problem associated with reduced therapeutic possibilities. Antimicrobial carbapenems are considered the last line of defense against multidrug-resistant Gram-negative bacteria. Unfortunately, an increase of carbapenem resistance due to the production of Klebsiellapneumoniae carbapenemase (KPC) enzymes has been observed. In this study we describe the ability of E. coli to produce carbapenemase enzymes based on the results of the combination disc assay with boronic acid performed according to guidelines established by the European Community on Antimicrobial Susceptibility Testing (EUCAST) and the biochemical Carba NP test. Moreover, we evaluated the presence of genes responsible for the production of carbapenemases (bla KPC, bla VIM, bla IMP, bla OXA-48) and genes encoding other β-lactamases (bla SHV, bla TEM, bla CTX-M) among E. coli isolate. The tested isolate of E. coli that possessed the bla KPC-3 and bla TEM-34 genes was identified. The tested strain exhibited susceptibility to colistin (0.38 μg/mL) and tigecycline (1 μg/mL). This is the first detection of bla KPC-3 in an E. coli ST479 in Poland. PMID:26339599

Background Klebsiellapneumoniae (KP) expressing hypermucoviscosity phenotype (HV-KP) has abundant capsular polysaccharide (CPS) and is capable of causing invasive syndrome. Sodium salicylate (SAL) reduces the production of CPS. The study was aimed to investigate the relationship between aspirin usage and KP-mediated invasive syndrome and the effect of SAL on HV-KP. Methods Patients with community-acquired KP bacteraemia were prospectively enrolled. KP-M1, a serotype-K1 HV-KP clinical isolate, was used in the following experiments: CPS production, HV-KP phenotype, and the effect of SAL on neutrophils phagocytosis. The effect of oral aspirin intake on the leukocyte bactericidal activity was evaluated. Results Patients infected by HV-KP and diabetic patients with poor glycemic control were at an increased risk for invasive syndrome (p

Background In many Asian countries, Klebsiellapneumoniae (KP) is the second pathogen responsible for community-acquired pneumonia. Yet, very little is known about KP etiology in ALRI in Cambodia, a country that has one of the weakest medical infrastructures in the region. We present here the first clinico-radiological description of KP community-acquired ALRI in hospitalized Cambodian patients. Methods Through ALRI surveillance in two provincial hospitals, KP was isolated from sputum and blood cultures, and identified by API20E gallery from patients ≥ 5 years-old with fever and respiratory symptoms onset ≤14 days. Antibiotics susceptibility testing was provided systematically to clinicians when bacteria were isolated. We collected patients' clinical, radiological and microbiological data and their outcome 3 months after discharge. We also compared KP-related with other bacteria-related ALRI to determine risk factors for KP infection. Results From April 2007 to December 2009, 2315 ALRI patients ≥ 5 years-old were enrolled including 587 whose bacterial etiology could be assigned. Of these, 47 (8.0%) had KP infection; their median age was 55 years and 68.1% were females. Reported prior medication was high (42.5%). Patients' chest radiographs showed pneumonia (61.3% including 39% that were necrotizing), preexisting parenchyma lesions (29.5%) and pleural effusions alone (4.5%) and normal parenchyma (4.5%). Five patients had severe conditions on admission and one patient died during hospitalization. Of the 39 patients that were hospital discharged, 14 died including 12 within 1 month after discharge. Only 13 patients (28%) received an appropriate antibiotherapy. Extended-spectrum beta-lactamases (ESBL) - producing strains were found in 8 (17.0%) patients. Female gender (Odds ratio (OR) 2.1; p = 0.04) and diabetes mellitus (OR 3.1; p = 0.03) were independent risk factors for KP-related ALRI. Conclusions KP ALRI in Cambodia has high fatality rate, are more

Hypervirulent Klebsiellapneumoniae isolates of capsular serotype K2 (hvKP-K2) that cause community-acquired invasive infections represent several unrelated clones, which all belong to phylogenetic group KpI. These clones can be recognized using multilocus sequence typing and genomic analyses, but no rapid method currently exists to differentiate them. In this work, a multiplex PCR assay was developed to identify three hvKP-K2 groups: (i) sequence type (ST)86; (ii) ST380 and ST679 (i.e. clonal group 380); and (iii) ST65 and ST375. A specific genetic marker, Kp50233, allowing K. pneumoniae sensu stricto (corresponding to phylogroup KpI) to be distinguished from closely related species, was included in the assay. This PCR assay will be useful in better defining the epidemiology and clinical features of emerging virulent K. pneumoniae clones. PMID:25261063

Klebsiellapneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats. PMID:26100894

Klebsiellapneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats. PMID:26100894

CTX-M extended-spectrum β-lactamase (ESBL)–producing Klebsiellapneumoniae isolates are infrequently reported in the United States. In this study, we analyzed nonduplicate ESBL-producing K. pneumoniae and Escherichia coli clinical isolates collected during 2005–2012 at a tertiary care medical center in suburban New York City, USA, for the presence of blaCTX-M, blaSHV, blaTEM, and blaKPC genes. Despite a high prevalence of blaCTX-M genes in ESBL-producing E. coli since 2005, blaCTX-M genes were not detected in K. pneumoniae until 2009. The prevalence of CTX-M–producing K. pneumoniae increased significantly over time from 1.7% during 2005–2009 to 26.4% during 2010–2012 (p<0.0001). CTX-M-15 was the dominant CTX-M genotype. Pulsed-field gel electrophoresis and multilocus sequence typing revealed high genetic heterogeneities in CTX-M–producing K. pneumoniae isolates. This study demonstrates the recent emergence and polyclonal spread of multidrug resistant CTX-M–producing K. pneumoniae isolates among patients in a hospital setting in the United States. PMID:24188126

One representative recombinant clone encoding Klebsiellapneumoniae O5-antigen lipopolysaccharide (LPS) was found upon screening for serum resistance in a cosmid-based genomic library of K. pneumoniae KT769 (O5:K57) introduced into Escherichia coli DH5α. A total of eight open reading frames (wbO5 gene cluster) were necessary to produce K. pneumoniae O5-antigen LPS in E. coli K-12. The enzymatic activities proposed for the wbO5 gene cluster are in agreement with the activities proposed for the biosynthesis of K. pneumoniae O5-antigen LPS. Using the complete DNA sequence of the K. pneumoniae wbO5 gene cluster, we obtained (by single or double recombination) genetically well-characterized mutants devoid only of this O5-antigen LPS. Finally, using these O5− mutants and the corresponding wild-type strains or complemented mutants with the wbO5 gene cluster (O5+ strains), we found that the presence of K. pneumoniae O5-antigen LPS is essential for some pathogenic features like serum resistance, adhesion to uroepithelial cells, and colonization (experimental infections) of the urinary tract in rats. PMID:10768928

The treatment of Klebsiellapneumoniae, particularly extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae, is currently a great challenge. Photodynamic antimicrobial chemotherapy is a promising approach for killing antibiotic-resistant bacteria. The aim of this study was to evaluate the capacity of 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL) in the presence of white light to cause photodynamic inactivation (PDI) of K. pneumoniae planktonic and biofilm cells. In the presence of white light, 5-ALA and MAL inactivated planktonic cells in a concentration-dependent manner. Biofilms were also sensitive to 5-ALA and MAL-mediated PDI. The mechanisms by which 5-ALA and MAL caused PDI of ESBL-producing K. pneumonia were also investigated. Exposure of K. pneumonia to light in the presence of either 5-ALA or MAL induced cleavage of genomic DNA and the rapid release of intracellular biopolymers. Intensely denatured cytoplasmic contents and aggregated ribosomes were also detected by transmission electron microscopy. Scanning electron microscopy showed that PDI of biofilms caused aggregated bacteria to detach and that the bacterial cell envelope was damaged. This study provides insights into 5-ALA and MAL-mediated PDI of ESBL-producing K. pneumoniae. PMID:26886586

Background and Objective: The production of carbapenemases especially Klebsiellapneumoniae carbapenemase (KPC) is the most important mechanism of enzymatic resistance in isolated Enterobacteriaceae such as K. pneumoniae . The purpose of this study was detected of the carbapenemase producer K. pneumoniae strains with phenotypic and genotypic methods. Method: Out of 800 strains, 270 K. pneumoniae strains (33.7%), were obtained. Antibiotic susceptibility test was performed by disk diffusion method in accordance with CLSI guidelines. Carbapenem resistant strains were identified by the Modified Hodge Test based on CLSI instruction and PCR for surveying the presence of bla -KPC gene. Results: A total 270 K. pneumoniae strains were collected. Antibiotic susceptibility test results showed the highest and lowest resistance was related to piperacillin (60.6%) and carbapenems (14.6%) respectively. 80.5% (33 of 41) isolates were positive by MHT, but all of them (100%) were negative for amplification of the bla -KPC gene in the PCR method. Conclusion: The MHT was an appropriate method for approving carbapenemase production. Moreover, a laboratory could accept the carbapenemase production with PCR method for the bla-KPC gene, which has the additional profit of validating which KPC is present. PMID:26351485

Objectives: To identify the mechanism of in vivo development of carbapenem resistance in Klebsiellapneumoniae.Methods: Seven sequential isolates of K. pneumoniae were obtained from twin infants with pneumonia. Antimicrobial susceptibility testing was performed by agar dilution method. Carbapenemases including KPC and MβL were initially screened using phenotypic methods, and carbapenemase-encoding genes were identified by polymerase chain reaction and amplicon sequencing. Plasmids of all clinical isolates and the conjugants of resistant isolates were estimated by S1 pulsed-field gel electrophoresis (PFGE). Molecular typing were conducted by PFGE of XbaI-digested genomic DNA and multilocus sequence typing.Results: For old brother, the first and third isolates were susceptible to meropenem, whereas the second and fourth isolates were resistant (MICs 16 mg/L). The first and second isolates from the young brother were susceptible to meropenem whereas the third isolate was resistant. All the resistant isolates produced NDM-1 metallo-β-lactamase. PFGE of XbaI-digested DNA revealed almost identical patterns with similarity indices of above 92% for all the seven isolates. All the isolates had the same sequence type named sequence type 37 (ST37).Conclusion: To our knowledge, this is the first documented case of development of carbapenem resistance in vivo mediated by NDM-1 metallo-β-lactamase in K. pneumoniae during treatment of pneumonia with meropenem. PMID:26733952

With the increasing use of broad-spectrum antibacterial agents, the increase in various drug-resistant bacterial strains has become a concern in recent years. Especially, the development of drug-resistance by Enterobacteriaceae which significantly affects therapy and prognosis in sepsis and lower gastrointestinal post-operative infection. The extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae strains isolated in the Surveillance Program of Bacterial Resistance in Kinki region of Japan (SBRK) were supplied between November 2000 and March 2003. The susceptibilities of them to 16 kinds of antimicrobial agents were investigated. The number of them was 48 strains consisting of 36 Escherichia coli strains (75%) and 12 Klebsiellapneumoniae strains (25%). Our focus was on carbapenem and the new quinolone antibacterial agents. Among the 16 major antibacterial agents examined, carbapenem had low MIC50/90 values. Meropenem had a MIC50/90 of 0.03/0.06microg/ml, followed by biapenem (0.12/0.5), imipenem (0.25/0.5) and panipenem (0.25/0.5). Among cephem, ceftazidime had the lowest MIC50 at 4 microg/ml. All four of the cephem agents had a MIC90 of greater than 128microg/ml. Among beta-lactamase inhibitors, tazobactam/piperacillin had the lowest MIC50 at 4 microg/ml, and sulbactam/cefoperazone had a MIC50 of 32 microg/ml. Among the new quinolones, prulifloxacin had the lowest MIC50 at 1 microg/ml, and the other drugs had a MIC50 of 2 microg/ml. The resistance rate of ciprofloxacin was 61.1% in E. coli and 16.6% in K. pneumoniae. Comparison of drug-sensitivity to cephem by ESBL-gene type revealed that cefpirome, cefepime and cefozopran had higher MIC50/90 values against the CTX-M group with a MIC50 of greater than 128microg/ml. Ceftazidime and aztreonam had higher MIC50/90 values against the TEM/SHV group than those against the CTX-M group. In the CTX-M group, the MIC50 was 4 and 16microg/ml, respectively. PMID:15847220

Most Klebsiellapneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is

Carriers of carbapenem-resistant Klebsiellapneumoniae (CRKP) are increasingly recognised through active surveillance in much of the world. We studied incidence, aetiology and predictors of bloodstream infections (BSI) among such carriers. Via a retrospective cohort study conducted in a tertiary care teaching hospital, we examined occurrence of BSI within 45 days of CRKP carrier detection. Three nested case-control studies were conducted to analyse parameters associated with all-cause (ALL), Gram-negative rod (GNR) and CRKP BSI. Cases and controls were compared with respect to demographics, clinical parameters and recent receipt of antibiotics. A total of 431 patients were identified as CRKP carriers (28% by clinical culture, 72% by rectal surveillance), mean age was 75.2 years. Twenty percent of the patients (n = 85) developed BSI, of them 80% (n = 68) with GNR. Of 83 GNR isolates, 58 (70%) were Enterobacteriaceae, of which 19 were CRKP and 20 were extended-spectrum β-lactamase (ESBL) producers (23% and 24% of total GNR, respectively); 29% of the GNR isolates were nonfermenters (14.5% Pseudomonas aeruginosa, 14.5% Acinetobacter baumannii). Mechanical ventilation predicted ALL BSI (p = 0.04), whereas Clostridium difficile-associated diarrhoea predicted GNR BSI (p = 0.04). Receipt of broad-spectrum antibiotics (piperacillin-tazobactam, amikacin, imipenem) was significantly associated with ALL BSI or GNR BSI. No exposure independently predicted CRKP BSI. We conclude that patients detected as CRKP carriers are at high risk for BSI within 45 days of detection, primarily with multidrug-resistant GNR. Lack of predictive factors differentiating between pathogens and associated high mortality raises once more the dilemma regarding the appropriate empiric therapy for CRKP carriers who develop severe sepsis. PMID:25636924

Background Animal models are useful to evaluate the efficacy of antimicrobials in experimental sepsis. Aim To elucidate the steps of producing an experimental model for the treatment of extended-spectrum beta-lactamase (ESBL)-producing Klebsiellapneumoniae sepsis Methods Several ESBL inoculums ranging from 1.5x109 colony-forming units per milliliter (CFU/mL) to 2.0x1010CFU/mL were administered by peritoneal injection in adults Wistar rats. Outcomes and microbiological data of quantitative peritoneal and blood cultures were observed in untreated animals. Animals which received 2.0x1010CFU/mL inoculums were treated with single meropenem dose (30mg/kg) after one hour and those which received 1.0x1010CFU/mL inoculums were treated immediately with three doses of meropenem 50 mg/kg. Outcomes were observed for 24 hours after inoculation. Results Solutions with 1.5 x109 and 6.0x109 CFU/mL were not lethal within 24 hours. Inoculums of 1.0x1010CFU/mL were lethal in 80% and solutions with 2.0x1010 CFU/mL were lethal in 100% of animals. ESBL lethal sepsis (1.0x1010CFU/mL) was treated immediately with 50 mg/kg of meropenem every eight hours for 24 hours and presented 40% mortality compared with 80% mortality of the control group (p=0.033). Quantitative cultures of peritoneal fluid presented 104CFU/mL or less for treated animals compared to more than 105 for untreated animals (p=0.001). Conclusion Inoculums of 1.0x1010CFU/mL achieved the best results to study a model of lethal sepsis and this model of treatment of carbapenem-susceptible Enterobacteriaceae can serve as control to further evaluation of treatment of carbapenemase-producing Enterobacteriaceae models. PMID:25184764

Polymyxins are reserved for salvage therapy of infections caused by carbapenem-resistant Klebsiellapneumoniae (CRKP). Though synergy has been demonstrated for the combination of polymyxins with carbapenems or tigecycline, in vitro synergy tests are nonstandardized, and the clinical effect of synergy remains unclear. This study describes outcomes for patients with CRKP infections who were treated with polymyxin B monotherapy. We retrospectively reviewed the medical records of patients with CRKP infections who received polymyxin B monotherapy from 2007 to 2011. Clinical, microbiology, and antimicrobial treatment data were collected. Risk factors for treatment failure were identified by logistic regression. Forty patients were included in the analysis. Twenty-nine of 40 (73%) patients achieved clinical cure as defined by clinician-documented improvement in signs and symptoms of infections, and 17/32 (53%) patients with follow-up culture data achieved microbiological cure. End-of-treatment mortality was 10%, and 30-day mortality was 28%. In a multivariate analysis, baseline renal insufficiency was associated with a 6.0-fold increase in clinical failure after adjusting for septic shock (odds ratio [OR] = 6.0; 95% confidence interval [CI] = 1.22 to 29.59). Breakthrough infections with organisms intrinsically resistant to polymyxins occurred in 3 patients during the treatment. Eighteen of 40 (45%) patients developed a new CRKP infection a median of 23 days after initial polymyxin B treatment, and 3 of these 18 infections were polymyxin resistant. The clinical cure rate achieved in this retrospective study was 73% of patients with CRKP infections treated with polymyxin B monotherapy. Baseline renal insufficiency was a risk factor for treatment failure after adjusting for septic shock. Breakthrough infections with organisms intrinsically resistant to polymyxin B and development of resistance to polymyxin B in subsequent CRKP isolates are of concern. PMID:23959321

During a national outbreak of carbapenem-resistant Klebsiellapneumoniae (CRKP) in Israel, we conducted a point prevalence survey to determine the extent of asymptomatic carriage. Subsequently, a retrospective case-control study was done, comparing carriers of CRKP with non-carriers, in order to detect risk factors for carriage. Oral, perianal and rectal swabs were obtained from all hospitalised eligible and consenting patients. Selective media for carbapenem-resistant Gram-negative bacteria were used and pulsed-field gel electrophoresis (PFGE) helped to determine clonal source. Culture was obtained from 298 patients. Sixteen (5.4%) were carriers of CRKP, with a higher carriage rate in medical and surgical wards. Only 18% of carriers were treated with any carbapenem prior to the survey. Five of the 16 carriers had a positive clinical specimen for CRKP, hence a clinical infection versus asymptomatic carriage ratio of 1:3. The rectum was the most sensitive site sampled, detecting 15/16 carriers, and the overall sensitivity of the method was 94% with a negative predictive value of 99.6%. In a multivariate analysis of risk factors for CRKP carriage, three variables were significantly related to carriage state: diaper use, longer duration of hospital stay and vancomycin use. PFGE demonstrated that all 16 isolates were identical, confirming clonal origin. A point prevalence survey performed at a single medical centre during an outbreak of CRKP demonstrated a carriage rate of 5.4%. The clonal origin of these isolates suggests that strict adherence to isolation procedure may contain this outbreak. PMID:19783067

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiellapneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs. PMID:26334306

Identifying disease vectors and pathogens is one of the key steps in controlling vector-borne diseases. This study investigated the possible role of house flies (Musca domestica) as vectors in the transmission of Klebsiellapneumoniae in Chaharmahal VA Bakhtiari and Isfahan provinces of Iran. House flies were captured from household kitchens, cattle farms, chicken farms, animal hospitals, human hospitals and slaughterhouses. Isolation of K. pneumoniae from external surfaces and guts of the flies was performed using MacConkey agar (MA) and thioglycollate broth (TGB). Identification of the isolates was performed with phenotypic techniques and polymerase chain reaction (PCR). A total of 600 house flies were sampled during the study period from different locations in four different seasons. Overall, 11.3% of the captured house flies were positive for K. pneumoniae. In Chaharmahal VA Bakhtiari province, the prevalence was 12.7%, while in Isfahan province, 10.0% of the sampled house flies were infected with K. pneumoniae. Season-wise, the highest prevalence of infections among the house flies was in summer. The organisms were highly resistant to ampicillin, amoxicillin, cefotaxime and piperacillin. A lowest level of resistance was observed for imipenem/cilastatin. The findings of this study demonstrated that house flies are potential vectors of antibiotic-resistant K. pneumoniae in Isfahan and Chaharmahal provinces, Iran. Control efforts for infections caused by this particular bacterium should take M. domestica into account. PMID:26876433

The increasing incidence of Klebsiellapneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K. pneumoniae Several monoclonal antibodies were isolated from phage display and hybridoma platforms by functional screening for opsonophagocytic killing activity. We further identified their common target antigen to be MrkA, a major protein in the type III fimbriae complex, and showed that these serotype-independent anti-MrkA antibodies reduced biofilm formation in vitro and conferred protection in multiple murine pneumonia models. Importantly, mice immunized with purified MrkA proteins also showed reduced bacterial burden following K. pneumoniae challenge. Taken together, these results support MrkA as a promising target for K. pneumoniae antibody therapeutics and vaccines. PMID:26768253

Legionella pneumophila, the agent of Legionnaires' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiellapneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×104 CFU per cm2 of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species. PMID:23185637

The emergence of Klebsiellapneumoniae multidrug-resistant strains paves the way to the re-introduction of colistin as a salvage therapy. However, recent planktonic studies have reported several cases of heteroresistance to this antimicrobial agent. The aim of this present work was to gain better understanding about the response of K. pneumoniae biofilms to colistin antibiotherapy and inspect the occurrence of heteroresistance in biofilm-derived cells. Biofilm formation and its susceptibility to colistin were evaluated through the determination of biofilm-cells viability. The profiling of planktonic and biofilm cell populations was conducted to assess the occurrence of heteroresistance. Colony morphology was further characterized in order to inspect the potential role of colistin in K. pneumoniae phenotypic differentiation. Results show that K. pneumoniae was susceptible to colistin in its planktonic form, but biofilms presented enhanced resistance. Population analysis profiles pointed out that K. pneumoniae manifest heteroresistance to colistin only when grown in biofilm arrangements, and it was possible to identify a resistant sub-population presenting a small colony morphology (diameter around 5 mm). To the best of our knowledge, this is the first report linking heteroresistance to biofilm formation and a morphological distinctive sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance in an apparently susceptible strain. PMID:27140200

Integrons are considered to play a significant role in the evolution and spread of antibiotic resistance genes. A total of 349 clinical isolates of Escherichia coli and Klebsiellapneumoniae were investigated for molecular characterization of integrons and betalactamases. Antimicrobial susceptibility testing was also performed as the Clinical and Laboratory Standards Institute (CLSI) guidelines. The frequency of extended spectrum betalactamases (ESBL) or metallo-betalactamases (MBL)-producing isolates, patient demographics, and the susceptibility to various antimicrobial agents were described. BlaCTX-M was the most frequently detected betalactamase in all isolates. Moreover, MBL producing K. pneumoniae carried blaIMP and blaVIM at 100 and 41·6%, respectively but no MBL-positive E. coli was detected. Class 1 integrons were more frequent among E. coli and K. pneumoniae isolates in comparison with class 2 integrons and the frequency of intI2 in K. pneumoniae was significantly higher than E. coli isolates. Five different resistance gene arrays were identified among class 1 integrons. Dihydrofolate reductase (dfrA) and aminoglycoside adenyltransferase (aad) gene cassettes were found to be predominant in the class 1 integrons. These results indicate that class 1 integrons are widespread among ESBL-producing isolates of K. pneumoniae and E. coli and appropriate surveillance and control measures are essential to prevent further dissemination of these elements among Enterobacteriaceae in our country. PMID:24571248

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiellapneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs. PMID:26334306

Strains with lac fused to each of the seven nif operons were isolated by two different methods. Repressing conditions prevented expression of all nif operons, whereas derepressing conditions led to the expression of all nif operons. No differences in Nif regulation were observed between Escherichia coli and Klebsiellapneumoniae with the same nif-lac fusions. Most derivatives of nif-lac fusion strains selected on lactose and NH4+ contained nif operator mutations. Some derivative contained deletions, which establishes that the direction of transcription of all seven nif operons is toward his Images PMID:7007318

Using time-kill methodology, we investigated the interactions of fosfomycin with meropenem or colistin or gentamicin against 17 genetically distinct Klebsiellapneumoniae clinical isolates carrying blaKPC-2. Synergy was observed with meropenem or colistin against 64.7 and 11.8% of tested isolates, while the combination with gentamicin resulted in indifference. All studied combinations showed improved bactericidal activity, compared to fosfomycin alone and prevented the development of fosfomycin resistance in 69.2, 53.8, and 81.8% of susceptible isolates, respectively. PMID:21321144

Thirty-two carbapenem-resistant Klebsiellapneumoniae isolates, representative of different resistance mechanisms and clonal lineages, were analyzed with the Pathogenica HAI BioDetection system, based on targeted next-generation sequencing (NGS) technology. With most strains, the system simultaneously yielded comprehensive information on relevant β-lactam resistance determinants and accurate discrimination of clonal lineages, in a shorter time frame and in a less labor-intensive manner than currently available methods for molecular epidemiology analysis. Results supported the usefulness of targeted NGS-based technologies for similar applications. PMID:24403299

Klebsiellapneumoniae strain MRSN2404 was isolated from the chronic wound of a soldier who had been wounded in Iraq in 2006. The strain displayed very high MICs of all aminoglycosides, including arbekacin. A gene encoding a novel 16S rRNA methyltransferase, now designated RmtH, was identified. RmtH had 64% identity with RmtB1 and RmtB2. rmtH was bracketed by two copies of ISCR2, which may have played a role in its mobilization. PMID:23478957

A CMY-2-producing capsular type K2 Klebsiellapneumoniae strain (TVGHKP93) with multidrug resistance was isolated from a recurrent liver abscess in a patient who also carried a CMY-2-producing Escherichia coli strain (TVGHEC01) in the stool. TVGHKP93 retained its high virulence compared with that of the isogenic strain (TVGHKP60) with wild-type resistance from the first liver abscess. Our conjugation experiment showed the successful transfer of the blaCMY-2-carrying plasmid from TVGHEC01 into TVGHKP60. The transconjugant showed both high virulence and the multidrug-resistant phenotype, as did TVGHKP93. PMID:25987637

The natural resistance of nonimmunized C57B1/6 mice to an intraperitoneal Klebsiellapneumoniae challenge was not significantly affected by prior continuous exposure to 20 ppm NO/sub 2/ for 4 days. In contrast, the acquired resistance of mice immunized just before and infected just after NO/sub 2/ exposure was seriously impaired. This could not be explained by the loss of appetite (about 30%) observed in NO/sub 2/ treated mice, for neither the natural nor acquired resistance of control air exposure mice given approximately 70% ad libitum food and water were significantly modified.

KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiellapneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family. PMID:20944219

Biochemical properties and antibiotic susceptibilities of 168 clinical isolates of Klebsiellapneumoniae were tested. On the basis of the indole reaction, 30 isolates (18%) were indole-positive and 138 isolates (82%) were indole-negative. A significant difference in antibiotic susceptibility was found in each of the two groups of isolates. Of the indole-negative isolates, 82.6% were susceptible to cefazolin at 6.25 microgram/ml or lower, while the indole-positive isolates varied in susceptibility to cefazolin. All of the indole-positive isolates were susceptible to chloramphenicol and tetracycline, but the indole-negative isolates varied in their susceptibility to these antibiotics. Whereas there was no relationship between indole production and susceptibility to cephalothin among the K. pneumoniae isolates studied, it appeared that there was a correlation between the indole reaction and susceptibility to cefazolin, chloramphenicol and tetracycline. PMID:350514

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na+-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium Klebsiellapneumoniae, and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na+-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na+-NQR from K. pneumoniae is fully resistant to either Ag+ or N-ethylmaleimide. All the Na+-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme. PMID:18300384

A 68-year-old male underwent a right hepatectomy, resection of the biliary convergence, and a left hepatic jejunostomy for a Klatskin tumour. The postoperative course was complicated by biliary abscesses with relapsing bloodstream infections due to Klebsiellapneumoniae carbapenemase (KPC)-producing Klebsiellapneumoniae (KPC-Kp). A 2-week course of combination antibiotic therapy failed to provide source control and the bacteraemia relapsed. Success was obtained with a regimen of tigecycline 100mg daily for 2 months, followed by tigecycline 50mg daily for 6 months, then 50mg every 48h for 3 months. No side effects were reported. PMID:25975648

Klebsiellapneumoniae can cause community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) is important for its virulence. Among 79 capsular (K) types discovered thus far, K57 is often associated with PLA. Here, we report the identification of a K57 variant. Cps gene locus sequencing revealed differences between the K57 reference strain 4425/51 (Ref-K57) and a variant, the PLA isolate A1142. While Ref-K57 cps contained orf13 encoding a putative acetyltransferase, the insertion of a putative transposase-encoding gene at this position was detected in A1142. This variation was detected in other K57 clinical strains. Biochemical analyses indicated that A1142 was deficient in CPS acetylation. Genetic replacement and complementation verified that orf13 was responsible for CPS acetylation. Acetylation increased CPS immunoreactivity to antiserum and enhanced K. pneumoniae induction of pro-inflammatory cytokines through JNK and MAPK signaling. While acetylation diminished the serum resistance of bacteria, it promoted adhesion to intestinal epithelial cells possibly via increasing production of type I fimbriae. In conclusion, acetylation-mediated capsular variation in K57 was observed. Capsular acetylation contributed to the variety and antigenic diversity of CPS, influenced its biological activities, and was involved in K. pneumoniae-host interactions. These findings have implications for vaccine design and pathogenicity of K. pneumoniae. PMID:27550826

Infection by bacteria carrying New Delhi metallo-β-lactamase 1 (NDM-1) is becoming a global health problem. We report a case of meningitis caused by NDM-1-producing Klebsiellapneumoniae, for which intrathecal administration of colistin was curative. A previously healthy 38-year-old Japanese man, who lived in Hyderabad, India, suddenly collapsed and was brought to a local hospital. He was diagnosed with subarachnoid hemorrhage and underwent emergency surgery which included partial skull removal. Approximately 1 month after surgery, he was repatriated to Japan and was admitted to our institution with information that he had been treated for multi-drug resistant Acinetobacter infection with colistin. A week after admission, he developed aspiration pneumonia due to NDM-1-producing K. pneumoniae, which was successfully treated by intravenous (IV) administration of colistin. Subsequently, he underwent a surgical procedure to repair his skull defect. He developed high-grade fever and altered mental status on postoperative day 2. NDM-1-producing K. pneumoniae was identified in the cerebrospinal fluid, establishing the diagnosis of meningitis. Although IV colistin was only partially effective, intrathecal colistin (10 mg daily by lumbar puncture for 14 days) successfully eradicated the meningitis. Because of economic globalization, NDM-1-producing bacteria may be brought to Japan by those who are repatriated after sustaining critical illnesses and being treated in foreign countries. This report may provide useful information on the treatment of central nervous system infection by NDM-1-producing bacteria. PMID:26683242

Infections due to carbapenem-resistant Klebsiellapneumoniae represent a growing problem nationally. In our study, we aimed to examine carbapenem-resistant K. pneumoniae with multiple resistance isolated in the intensive care unit of our hospital. Isolates were investigated for the presence of oxacillinase and metallo-beta lactamase genes with a view to determining the clonal relationship between the strains intensely over a short period. Strain identification was completed with conventional methods and automated identification kit. OXA-58, OXA-23, OXA-51, OXA-24 and OXA-48 and metallo-beta lactamase genes IPM, VIM, SPM, SIM, GIM and NDM-1 were investigated with PCR. For clonal relationships of carbapenem-resistant strains, the PFGE experiment was performed. While all of these carbapenem-resistant strains were positive for OXA-48, the resistant genes NDM-1, VIM, KPC, IPM, SPM, GIM, SIM, OXA-23, OXA-24, OXA-58 and OXA-51 were not observed. When molecular typing results were investigated, PFGE determined clonal distribution of three pulsotypes. However, it was observed that the strains intensified in a single clone and this was assessed as the outbreak isolate. The results of this study showed the primary enzyme responsible for carbapenem resistance in K. pneumoniae strains in our hospital is still OXA-48. To prevent the spread of carbapenem-resistant K. pneumoniae isolates, with epidemic potential, national-level monitoring and effective infection control precautions should be enforced. PMID:27031897

During 2013, ST278 Klebsiellapneumoniae with blaNDM-7 was isolated from the urine (KpN01) and rectum (KpN02) of a patient in Calgary, Canada. The same strain (KpN04) was subsequently isolated from another patient in the same unit. Interestingly, a carbapenem-susceptible K. pneumoniae ST278 (KpN06) was obtained 1 month later from the blood of the second patient. Next-generation sequencing (NGS) revealed that the loss of carbapenem-resistance in KpN06 was due to a 5-kb deletion on the blaNDM-7-harboring IncX3 plasmid. In addition, an IncFIB plasmid in KpN06 had a 27-kb deletion that removed genes encoding for heavy metal resistance. Phylogenetic analysis showed that the K. pneumoniae ST278 from patient 2 was likely a descendant of KpN02 and that KpN06 was a close progenitor of an environmental ST278. It is unclear whether KpN06 lost the blaNDM-7 gene in vivo. This study detailed the remarkable plasticity and speed of evolutionary changes in multidrug-resistant K. pneumoniae, demonstrating the highly recombinant nature of this species. It also highlights the ability of NGS to clarify molecular microevolutionary events within antibiotic-resistant organisms. PMID:27284091

Klebsiellapneumoniae can cause community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) is important for its virulence. Among 79 capsular (K) types discovered thus far, K57 is often associated with PLA. Here, we report the identification of a K57 variant. Cps gene locus sequencing revealed differences between the K57 reference strain 4425/51 (Ref-K57) and a variant, the PLA isolate A1142. While Ref-K57 cps contained orf13 encoding a putative acetyltransferase, the insertion of a putative transposase-encoding gene at this position was detected in A1142. This variation was detected in other K57 clinical strains. Biochemical analyses indicated that A1142 was deficient in CPS acetylation. Genetic replacement and complementation verified that orf13 was responsible for CPS acetylation. Acetylation increased CPS immunoreactivity to antiserum and enhanced K. pneumoniae induction of pro-inflammatory cytokines through JNK and MAPK signaling. While acetylation diminished the serum resistance of bacteria, it promoted adhesion to intestinal epithelial cells possibly via increasing production of type I fimbriae. In conclusion, acetylation-mediated capsular variation in K57 was observed. Capsular acetylation contributed to the variety and antigenic diversity of CPS, influenced its biological activities, and was involved in K. pneumoniae-host interactions. These findings have implications for vaccine design and pathogenicity of K. pneumoniae. PMID:27550826

Background Klebsiellapneumoniae is one of the most important pathogens responsible for nosocomial outbreaks worldwide. Epidemiological analyses are useful in determining the extent of an outbreak and in elucidating the sources and the spread of infections. The aim of this study was to investigate the epidemiological spread of K. pneumoniae strains using a MALDI-TOF MS approach. Methods Five hundred and thirty-five strains of K. pneumoniae were collected between January 2008 and March 2011 from hospitals in France and Algeria and were identified using MALDI-TOF. Antibiotic resistance patterns were investigated. Clinical and epidemiological data were recorded in an Excel file, including clustering obtained from the MSP dendrogram, and were analyzed using PASW Statistics software. Results Antibiotic susceptibility and phenotypic tests of the 535 isolates showed the presence of six resistance profiles distributed unequally between the two countries. The MSP dendrogram revealed five distinct clusters according to an arbitrary cut-off at the distance level of 500. Data mining analysis of the five clusters showed that K. pneumoniae strains isolated in Algerian hospitals were significantly associated with respiratory infections and the ESBL phenotype, whereas those from French hospitals were significantly associated with urinary tract infections and the wild-type phenotype. Conclusions MALDI-TOF was found to be a promising tool to identify and differentiate between K. pneumoniae strains according to their phenotypic properties and their epidemiological distribution. This is the first time that MALDI-TOF has been used as a rapid tool for typing K. pneumoniae clinical isolates. PMID:23620754

Background The rates of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates among Enterobacteriaceae isolates, particularly Klebsiellapneumoniae, have risen substantially worldwide. Methodology/Principal Findings To better understand the molecular mechanisms of drug resistance in K. pneumoniae, we analyzed the drug resistance determinants for K. pneumoniae isolates collected from the 306 Hospital, a tertiary-care hospital in Beijing, China, for the period of September 1, 2010-October 31, 2011. Drug susceptibility testing, PCR amplification and sequencing of the drug resistance determinants were performed. Conjugation experiments were conducted to examine the natural ability of drug resistance to disseminate among Enterobacteriaceae strains using a sodium azide-resistant Escherichia coli J53 strain as a recipient. Among the 223 consecutive non-repetitive K. pneumoniae isolates included in this study, 101 (45.3%) were extended-spectrum beta-lactamases (ESBLs) positive. The rates of MDR, XDR, and PDR isolates were 61.4% (n = 137), 22.0% (n = 49), and 1.8% (n = 4), respectively. Among the tested drug resistance-associated genes, the following ones were detected at relatively high rates blaCTX-M-10 (80, 35.9%), aacC2 (73, 32.7%), dhfr (62, 27.8%), qnrS (58, 26.0%), aacA4 (57, 25.6%), aadA1 (56, 25.1%). Results from conjugation experiments indicate that many of the drug resistance genes were transmissible. Conclusions/Significance Our data give a “snapshot” of the complex genetic background responsible for drug resistance in K. pneumoniae in China and demonstrate that a high degree of awareness and monitoring of those drug resistance determinants are urgently needed in order to better control the emergence and transmission of drug-resistant K. pneumoniae isolates in hospital settings. PMID:22860106

Kpp95, isolated on Klebsiellapneumoniae, is a bacteriophage with the morphology of T4-type phages and is capable of rapid lysis of host cells. Its double-stranded genomic DNA (ca. 175 kb, estimated by pulsed-field gel electrophoresis) can be cut only by restriction endonucleases with a cleavage site flanked either by A and T or by T, as tested, suggesting that it contains the modified derivative(s) of G and/or C. Over 26 protein bands were visualized upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virion proteins. N-terminal sequencing indicated that the most abundant band (46 kDa) is the major coat protein (gp23) which has been cleaved from a signal peptide likely with a length similar to that of T4. Phylogenetic analyses based on the sequences of the central region (263 amino acid residues) of gp23 and the full length of gp18 and gp19 placed Kpp95 among the pseudo-T-even subgroup, most closely related to the coliphage JS98. In addition to being able to lyse many extended-spectrum beta-lactamase strains of K. pneumoniae, Kpp95 can lyse Klebsiella oxytoca, Enterobacter agglomerans, and Serratia marcescens cells. Thus, Kpp95 deserves further studies for development as a component of a therapeutic cocktail, owing to its high efficiencies of host lysis plus extended host range. PMID:17337566

The factors responsible for colonization of the small intestine by enterotoxigenic coliform bacteria in Puerto Ricans with tropical sprue are unknown, but epidemiological observations have suggested that they may be related to an increased dietary intake of long-chain unsaturated fatty acids, particularly linoleic acid, which is known to exert an inhibitory effect on the growth of gram-positive organisms that normally comprise the flora of the small intestine. We have examined, by using a glucose-limited continuous-culture system, what effect this fatty acid exerts on the growth relationships of enteric gram-positive and coliform bacteria. In this system, colonization by an invading strain of Klebsiellapneumoniae was prevented by the presence of an established culture of Lactobacillus acidophilus, principally by virtue of a lowered pH of the medium that was incompatible with Klebsiella growth. However, when the population density of L. acidophilus was reduced by the presence of a sufficient concentration of linoleic acid, the invading K. pneumoniae successfully colonized the system and, once established, suppressed the growth of L. acidophilus. These observations indicate that, under the conditions of our chemostat, gram-positive enteric bacteria suppress coliform growth and that this effect is reversible by the presence of linoleic acid. It remains to be established, however, what pertinence these in vitro observations have to conditions within the intestinal tract of persons living in the tropics. PMID:811564

We investigated the clinical and microbiological epidemiology of AmpC plasmidic cephalosporinases (pAmpC) in Klebsiellapneumoniae strains resistant to ceftazidime, during a 3-year period (2007-2009). Among 1505 K. pneumoniae, 7 were pAmpC producers. Molecular characterization revealed the spread of a ST37 strain producing DHA-1 within intensive care units and the diffusion of the same plasmid among unrelated strains. PMID:22749243

The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiellapneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae. PMID:27147066

The recent emergence of carbapenemase-producing Enterobacteriaceae strains represents a major threat for hospitalized patients. We document the dissemination and control of carbapenemase-producing Klebsiellapneumoniae clones in a Greek hospital. During a 3-year study period (January 2009 to December 2011), carbapenemase-producing K. pneumoniae strains were isolated from clinical samples from 73 individual patients. Phenotyping and molecular testing confirmed that 52 patients were infected with K. pneumoniae carbapenemase 2 (KPC-2) producers, 12 were infected with VIM-1 producers, and the remaining 9 were infected with isolates producing both KPC-2 and VIM-1 enzymes. Twenty-eight of these clinical cases were characterized as imported health care associated, and 23 of these were attributed to KPC producers and 5 were attributed to KPC and VIM producers. The remaining 45 cases were deemed hospital acquired. In the second year of the study, intensified infection control intervention was implemented, followed by active surveillance and carrier isolation in the third year. The incidence of carbapenemase-producing K. pneumoniae patient cases decreased from 0.52/1,000 patient days in 2009 to 0.32/1,000 patient days in 2010 (P = 0.075). Following these additional infection control measures, the incidence fell to 0.21/1,000 patient days in 2011 and differed significantly from that in 2009 (P = 0.0028). Despite the fact that the imported cases of carbapenemase-producing K. pneumoniae were equally distributed over this 3-year period, the incidence of hospital-acquired cases decreased from 0.36/1,000 patient days in 2009 to 0.19/1,000 patient days in 2010 (P = 0.058) and to 0.1/1,000 patient days in 2011 (P = 0.0012). Our findings suggest that rigorous infection control measures and active surveillance can effectively reduce the incidence of secondary transmission due to KPC-producing pathogens. PMID:22649010

Background Oregano (Origanum vulgare), sage (Salvia officinalis), and thyme (Thymus vulgaris) are aromatic plants with ornamental, culinary, and phytotherapeutic use all over the world. In Europe, they are traditionally used in the southern countries, particularly in the Mediterranean region. The antimicrobial activities of the essential oils (EOs) derived from those plants have captured the attention of scientists as they could be used as alternatives to the increasing resistance of traditional antibiotics against pathogen infections. Therefore, significant interest in the cultivation of various aromatic and medicinal plants is recorded during the last years. However, to gain a proper and marketable chemotype various factors during the cultivation should be considered as the geographical morphology, climatic, and farming conditions. In this frame, we have studied the antimicrobial efficiency of the EOs from oregano, sage, and thyme cultivated under different conditions in a region of NE Greece in comparison to the data available in literature. Methods Plants were purchased from a certified supplier, planted, and cultivated in an experimental field under different conditions and harvested after 9 months. EOs were extracted by using a Clevenger apparatus and tested for their antibacterial properties (Minimum inhibitory concentration – MIC) against clinical isolates of multidrug resistant Escherichia coli (n=27), Klebsiella oxytoca (n=7), and Klebsiellapneumoniae (n=16) strains by using the broth microdilution assay. Results Our results showed that the most sensitive organism was K. oxytoca with a mean value of MIC of 0.9 µg/mL for oregano EOs and 8.1 µg/mL for thyme. The second most sensitive strain was K. pneumoniae with mean MIC values of 9.5 µg/mL for thyme and 73.5 µg/mL for oregano EOs. E. coli strains were among the most resistant to EOs antimicrobial action as the observed MICs were 24.8–28.6 µg/mL for thyme and above 125 µg/mL for thyme and sage

The in vitro and in vivo effectiveness of amikacin, cefepime, and imipenem was studied using a high inoculum of an extended-spectrum β-lactamase-producing Klebsiellapneumoniae strain. An in vitro susceptibility test at the standard inoculum predicted the in vivo outcome of amikacin or imipenem while it did not do so for cefepime due to the inoculum effect. PMID:11257049

KPC-producing Klebsiellapneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates. PMID:25734903

Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiellapneumoniae were experimentally determined through modified 5′ RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5′ UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5′ UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5′ UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization. PMID:22912590

In June 2010, a blaKPC-negative, ertapenem-resistant ST-258 Klebsiellapneumoniae strain was isolated from a patient in the Laniado Medical Center (LMC). Our aims were (i) to describe its molecular characteristics and resistance mechanisms and (ii) to assess whether the blaKPC-negative ST-258 K. pneumoniae clone spreads as efficiently as its KPC-producing isogenic strain. In a prospective study, surveillance of all ertapenem-resistant, carbapenemase-negative K. pneumoniae (ERCNKP) isolates was conducted from June 2010 to May 2011 at LMC (314 beds) and from July 2008 to December 2010 at the Tel Aviv Sourasky Medical Center (TASMC) (1,200 beds). Molecular typing was done by arbitrarily primed PCR, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). A total of 8 of 42 (19%) ERCNKP isolates in LMC and 1 of 32 (3.1%) in TASMC belonged to the ST-258 clone. These strains carried the blaCTX-M-2 or the blaCTX-M-25 extended-spectrum β-lactamase (ESBL) gene. Sequencing of the ompK genes showed a frameshift mutation in the ompK35 gene. The fate of the blaKPC-carrying plasmid, pKpQIL, was determined by S1 analysis and by PCR of the Tn4401 transposon, repA, and the truncated blaOXA-9. Plasmid analysis of the ERCNKP ST-258 isolates showed variability in plasmid composition and absence of the Tn4401 transposon and the pKpQIL plasmid. In addition, the ST-258 clone was identified in 35/35 (100%) of KPC-producing K. pneumoniae isolates but in none of 62 ertapenem-susceptible K. pneumoniae isolates collected in the two centers. Our results suggest that ERCNKP ST-258 evolved by loss of the blaKPC-carrying plasmid pKpQIL. ERCNKP ST-258 appears to have low epidemic potential. PMID:22814467

Background Klebsiellapneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044. Results In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism. Conclusions Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae. PMID:24885329

Objectives Observational studies have suggested that Escherichia coli sequence type (ST) 131 and Klebsiellapneumoniae ST258 have hyperendemic properties. This would be obvious from continuously high incidence and/or prevalence of carriage or infection with these bacteria in specific patient populations. Hyperendemicity could result from increased transmissibility, longer duration of infectiousness, and/or higher pathogenic potential as compared with other lineages of the same species. The aim of our research is to quantitatively estimate these critical parameters for E. coli ST131 and K. pneumoniae ST258, in order to investigate whether E. coli ST131 and K. pneumoniae ST258 are truly hyperendemic clones. Primary outcome measures A systematic literature search was performed to assess the evidence of transmissibility, duration of infectiousness, and pathogenicity for E. coli ST131 and K. pneumoniae ST258. Meta-regression was performed to quantify these characteristics. Results The systematic literature search yielded 639 articles, of which 19 data sources provided information on transmissibility (E. coli ST131 n=9; K. pneumoniae ST258 n=10)), 2 on duration of infectiousness (E. coli ST131 n=2), and 324 on pathogenicity (E. coli ST131 n=285; K. pneumoniae ST258 n=39). Available data on duration of carriage and on transmissibility were insufficient for quantitative assessment. In multivariable meta-regression E. coli isolates causing infection were associated with ST131, compared to isolates only causing colonisation, suggesting that E. coli ST131 can be considered more pathogenic than non-ST131 isolates. Date of isolation, location and resistance mechanism also influenced the prevalence of ST131. E. coli ST131 was 3.2 (95% CI 2.0 to 5.0) times more pathogenic than non-ST131. For K. pneumoniae ST258 there were not enough data for meta-regression assessing the influence of colonisation versus infection on ST258 prevalence. Conclusions With the currently available data

Colistin, tigecycline, levofloxacin, tobramycin, and rifampin alone and in combination with doripenem were investigated for their in vitro activities and postantibiotic effects (PAEs) on Klebsiellapneumoniae. The in vitro activities of tested antibiotics in combination with doripenem were determined using a microbroth checkerboard technique. To determine the PAEs, K. pneumoniae strains in the logarithmic phase of growth were exposed for 1 h to antibiotics, alone and in combination. Recovery periods of test cultures were evaluated using viable counting after centrifugation. Colistin, tobramycin, and levofloxacin produced strong PAEs ranging from 2.71 to 4.23 h, from 1.31 to 3.82 h, and from 1.35 to 4.72, respectively, in a concentration-dependent manner. Tigecycline and rifampin displayed modest PAEs ranging from 1.18 h to 1.55 h and 0.92 to 1.19, respectively. Because it is a beta-lactam, PAEs were not exactly induced by doripenem (ranging from 0.10 to 0.18 h). In combination, doripenem scarcely changed the duration of PAE of each tested antibiotic alone. The findings of this study may have important implications for the timing of doses during K. pneumoniae therapy with tested antibiotics. PMID:25530961

Analysis of the genome of Klebsiellapneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5'-TTN-3' was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem. PMID:27531594

Klebsiellapneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity. PMID:22312293

Caveolin-1 (Cav1) is a structural protein of caveolae. Although Cav1 is associated with certain bacterial infections, it is unknown whether Cav1 is involved in host immunity against Klebsiellapneumoniae, the third most commonly isolated microorganism from bacterial sepsis patients. Here, we showed that cav1 knockout mice succumbed to K. pneumoniae infection with markedly decreased survival rates, increased bacterial burdens, intensified tissue injury, hyperactive proinflammatory cytokines, and systemic bacterial dissemination as compared with WT mice. Knocking down Cav1 by a dominant negative approach in lung epithelial MLE-12 cells resulted in similar outcomes (decreased bacterial clearance and increased proinflammatory cytokine production). Furthermore, we revealed that STAT5 influences the GSK3β-β-catenin-Akt pathway, which contributes to the intensive inflammatory response and rapid infection dissemination seen in Cav1 deficiency. Collectively, our findings indicate that Cav1 may offer resistance to K. pneumoniae infection, by affecting both systemic and local production of proinflammatory cytokines via the actions of STAT5 and the GSK3β-β-catenin-Akt pathway. PMID:22678904

Analysis of the genome of Klebsiellapneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5′-TTN-3′ was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem. PMID:27531594

In Klebsiellapneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD(+) is exhausted. Here we show that NAD(+) regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD(+)-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD(+). When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD(+) enhanced the activity of ALD4. More interestingly, the growth rates of all NAD(+)-regenerating strains were prolonged in comparison with the control, indicating that NAD(+) regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD(+) availability but also provides a method for regulating the dha regulon. PMID:23794046

Oncocins and apidaecins are short proline-rich antimicrobial peptides (PrAMPs) representing novel antibiotic drug lead compounds that kill bacteria after internalization and inhibition of intracellular targets (e.g. 70S ribosome and DnaK). Oncocin Onc72 is highly active against Gram-negative bacteria in vitro and in vivo protecting mice in systemic infection models with Escherichia coli and KPC-producing Klebsiellapneumoniae. Here we studied its efficacy in a murine thigh infection model using meropenem as antibiotic comparator that had a 44-fold higher molar in vitro activity than Onc72. Male CD1 mice were rendered neutropenic using cyclophosphamide for four days before intramuscular infection with K. pneumoniae ATCC 43816. After 75 min oncocin Onc72 or the antibiotic comparator meropenem were administered subcutaneously with 100 mg (43 µmol) and 25 mg (65 µmol) per kg of body weight, respectively, six times every 75 min. Onc72 and meropenem administered subcutaneously reduced the thigh tissue burden of K. pneumoniae ATCC 43816 in neutropenic mice significantly by 4.14 and 4.65 a log10 cfu/g, respectively. The bacterial counts were ∼0.5 and ∼1 log10 below the pre-treatment burden, respectively, indicating bactericidal effects for both compounds. Thus, Onc72 was as efficacious as meropenem in vivo despite its much lower in vitro activity determined according to CLSI standard antimicrobial activity tests. PMID:25968331

NifQ- and Mol- mutants of Klebsiellapneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high. PMID:3905765

Resistance to the antibacterial antifolate trimethoprim (TMP) is increasing in members of the family Enterobacteriaceae, driving the design of next-generation antifolates effective against these Gram-negative pathogens. The propargyl-linked antifolates are potent inhibitors of dihydrofolate reductases (DHFR) from several TMP-sensitive and -resistant species, including Klebsiellapneumoniae. Recently, we have determined that these antifolates inhibit the growth of strains of K. pneumoniae, some with MIC values of 1 μg/ml. In order to further the design of potent and selective antifolates against members of the Enterobacteriaceae, we determined the first crystal structures of K. pneumoniae DHFR bound to two of the propargyl-linked antifolates. These structures highlight that interactions with Leu 28, Ile 50, Ile 94, and Leu 54 are necessary for potency; comparison with structures of human DHFR bound to the same inhibitors reveal differences in residues (N64E, P61G, F31L, and V115I) and loop conformations (residues 49 to 53) that may be exploited for selectivity. PMID:25288083

Background The increasing number of infections caused by strains of Klebsiellapneumoniae that are resistant to multiple antibiotics has developed into a major medical problem worldwide. The development of next-generation sequencing technologies now permits rapid sequencing of many K. pneumoniae isolates, but sequence information alone does not provide important structural and operational information for its genome. Results Here we take a systems biology approach to annotate the K. pneumoniae MGH 78578 genome at the structural and operational levels. Through the acquisition and simultaneous analysis of multiple sample-matched –omics data sets from two growth conditions, we detected 2677, 1227, and 1066 binding sites for RNA polymerase, RpoD, and RpoS, respectively, 3660 RNA polymerase-guided transcript segments, and 3585 transcription start sites throughout the genome. Moreover, analysis of the transcription start site data identified 83 probable leaderless mRNAs, while analysis of unannotated transcripts suggested the presence of 119 putative open reading frames, 15 small RNAs, and 185 antisense transcripts that are not currently annotated. Conclusions These findings highlight the strengths of systems biology approaches to the refinement of sequence-based annotations, and to provide new insight into fundamental genome-level biology for this important human pathogen. PMID:23194155

3-Hydroxypropionic acid (3-HP) is a commercially valuable platform compound. Klebsiellapneumoniae has been concerned as an appropriate host for 3-HP production because of its robust capacity to metabolize glycerol. Glycerol conversion to 3-HP in K. pneumoniae comprises two successive reactions: glycerol dehydratase catalyzes glycerol to 3-hydroxypropionaldehyde (3-HPA); aldehyde dehydrogenase catalyzes 3-HPA to 3-HP. Previous studies focusing on inducible expression of aldehyde dehydrogenase have shown defects of high cost of inducer and low catalytic activity due to inclusion body. Here we show a different strategy that a native promoter in the host K. pneumoniae was used to drive the heterologous expression of aldehyde dehydrogenase gene ald4 from Saccharomyces cerevisiae. The 3-HP yield of the recombinant reached a peak of 4.23 g/L at log phase, but it decreased during later period of fermentation. Except the validation of high activity of ald4, particularly, the 3-HP formation was uncovered to be closely coupled with cell division, and the lacking of NAD and ATP at latter fermentation phase became the bottleneck for cell growth and 3-HP accumulation. Furthermore, 3-HP is postulated to be converted to 3-HPA via feedback inhibition or other metabolite via unknown mechanism. Since glycerol dissimilation is a common mechanism in a variety of bacteria, the expression strategy using native promoter and implications may provide significant insight into the metabolic engineering for 3-HP production. PMID:23997342

Extended-spectrum -lactamase producing Klebsiellapneumoniae have emerged as one of the major nosocomial pathogens. Between July and September 2012, a CTX-M-15 producing K. pneumoniae caused an outbreak in a university hospital in the Netherlands. The outbreak isolates were characterized and assigned to a novel sequence type (ST1427). An epidemiological link between affected patients was supported by patient contact tracing and whole-genome phylogenetic analysis. Intra-strain polymorphism was detected among multiple isolates obtained from different body sites of the index patient, which may relate to antibiotic treatment and/or host adaptation. Environmental contamination caused by the outbreak clone was found in the patient rooms even on medical equipment. The novel clone was not closely related to any known endemic/epidemic clone, but carried a set of a plasmid-borne resistance genes [blaCTX−M−15, blaTEM−1, blaOXA−1, aac(6′)-Ib-cr, qnrB1, tetA(A), aac(3)-II]. Analysis of its virulence factors revealed a previously uncharacterized capsular biosynthesis region and two uncharacterized fimbriae gene clusters, and suggested that the new clone was not hypervirulent. To our knowledge, this is the first outbreak report of K. pneumoniae ST1427, and our study could be of help to understand the features of this newly emerging clone. PMID:26617589

NifQ/sup -/ and Mol/sup -/ mutants of Klebsiellapneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ/sup -/ and Mol/sup -/ mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. The data show that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. This study suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.

BACKGROUND: It has been well documented in several studies that many immunologic parameters are altered in experimental animals and human subjects who have flown in space. However, it is not fully known whether these immunologic changes could result in increased susceptibility to infection. Hindlimb (antiorthostatic) unloading of rodents has been used successfully to simulate some of the effects of spaceflight on physiologic systems. OBJECTIVE: The objective of this study was to determine the effect of hindlimb unloading on the outcome of Klebsiellapneumoniae infection in mice. METHODS: Hindlimb-unloaded, hindlimb-restrained, and control mice were intraperitoneally infected with one 50% lethal dose of K pneumoniae 2 days after suspension. Mortality and bacterial load in several organs were compared among the groups. RESULTS: Unloaded mice showed significantly increased mortality and reduced mean time to death compared with that seen in the control groups. Kinetics of bacterial growth with smaller infective doses revealed that control mice were able to clear bacteria from the organs after 30 hours. In contrast, unloaded mice had continued bacterial growth at the same time point. CONCLUSION: The results of this study suggest that hindlimb unloading might enhance the dissemination of K pneumoniae, leading to increased mortality. The complex physiologic changes observed during hindlimb unloading, including stress, have a key role in the pathophysiology of this infection.

Klebsiellapneumoniae strains producing K. pneumoniae carbapenemase (KPC) cause serious infections in debilitated and immunocompromised patients and are associated with prolonged hospital stays and increased mortality rates. Daptomycin is a lipopeptide used against Staphylococcus aureus infection and considered inactive against Gram-negative bacteria. We investigated the effectiveness of a daptomycin-meropenem combination by synergy kill curve and a pharmacokinetic/pharmacodynamic model. The combination may represent a novel therapeutic strategy against infections caused by KPC-producing K. pneumoniae strains. PMID:27216067

Introduction Community acquired K. pneumoniaepneumonia is still common in Asia and is reportedly associated with alcohol use. Oropharyngeal carriage of K. pneumoniae could potentially play a role in the pathogenesis of K. pneumoniaepneumonia. However, little is known regarding K. pneumoniae oropharyngeal carriage rates and risk factors. This population-based cross-sectional study explores the association of a variety of demographic and socioeconomic factors, as well as alcohol consumption with oropharyngeal carriage of K. pneumoniae in Vietnam. Methods and Findings 1029 subjects were selected randomly from age, sex, and urban and rural strata. An additional 613 adult men from a rural environment were recruited and analyzed separately to determine the effects of alcohol consumption. Demographic, socioeconomic, and oropharyngeal carriage data was acquired for each subject. The overall carriage rate of K. pneumoniae was 14.1% (145/1029, 95% CI 12.0%–16.2%). By stepwise logistic regression, K. pneumoniae carriage was found to be independently associated with age (OR 1.03, 95% CI 1.02–1.04), smoking (OR 1.9, 95% CI 1.3–2.9), rural living location (OR 1.6, 95% CI 1.1–2.4), and level of weekly alcohol consumption (OR 1.7, 95% CI 1.04–2.8). Conclusion Moderate to heavy weekly alcohol consumption, old age, smoking, and living in a rural location are all found to be associated with an increased risk of K. pneumoniae carriage in Vietnamese communities. Whether K. pneumoniae carriage is a risk factor for pneumonia needs to be elucidated. PMID:24667800

Objective: Extended-spectrum β-lactamase producing K. pneumoniae is a serious threat to the patients. This manuscript shows the comparison of phenotypic characterization methods used for ESBL K. pneumoniae and frequency distribution of these isolates in various clinical samples. Methodology: Eleven different types of pathological samples collected on various time intervals were analyzed. K. pneumoniae were identified with API 20E system (bioMerieux) and initial screening of ESBL K. pneumoniae was performed using the ceftazidime antimicrobial disc. Double-disc synergy test (DDST) and CLSI confirmatory test were compared for the phenotypic detection of ESBL K. pneumoniae. Results: A total number of 214 ESBL producing K. pneumoniae were isolated from various clinical samples. Frequency distribution of ESBL producing K. pneumoniae was found to be highest among blood 117 (54.7%) and urine 46 (21.5%) samples. Data regarding the use of various interventions among these patients showed most common presence of intravenous line 209 (97.7%) and urinary catheters 46 (21.5%). Comparison of DDST and CLSI confirmatory test showed that the DDST detected 145 (67.8%) isolates while 213 (99.5%) ESBL K. pneumoniae were characterized by CLSI confirmatory test. Conclusion: The use of CLSI confirmatory test is very efficient in the early detection of ESBL K. pneumoniae especially when the facilities for molecular characterization are not available. PMID:24353625

Adherence to host cells by a bacterial pathogen is a critical step for establishment of infection. It will contribute greatly to the understanding of bacterial pathogenesis by studying the biological force between a single pair of pathogen and host cell. In our experiment, we use a calibrated optical tweezers system to detach a single Klebsiellapneumoniae, the pathogen, from collagen, the host. By gradually increasing the laser power of the optical tweezers until the Klebsiellapneumoniae is detached from the collagen, we obtain the magnitude of the adhesive force between them. This happens when the adhesive force is barely equal to the trapping force provided by the optical tweezers at that specific laser power. This study is important because Klebsiellapneumoniae is an opportunistic pathogen which causes suppurative lesions, urinary and respiratory tract infections. It has been proved that type 3 fimbrial adhesin (mrkD) is strongly associated with the adherence of Klebsiellapneumoniae. Besides, four polymorphic mrkD alleles: namely, mrkDv1, v2, v3, and v4, are typed by using RFLP. In order to investigate the relationship between the structure and the function for each of these variants, DNA fragments encoding the major fimbrial proteins mrkA, mrkB, mrkC are expressed together with any of the four mrkD adhesins in E. coli JM109. Our study shows that the E. coli strain carrying the mrkDv3 fimbriae has the strongest binding activity. This suggests that mrkDv3 is a key factor that enhances the adherence of KlebsiellaPneumoniae to human body.

The in-vivo activity of imipenem against VIM-1-producing Klebsiellapneumoniae (VPKP) was assessed in a thigh infection model in neutropenic mice. Animals were infected with three VPKP isolates (imipenem MICs 2, 4 and 32 mg/L, respectively) and a susceptible clinical isolate (MIC 0.125 mg/L) that did not produce any beta-lactamase with broad-spectrum activity. Bacterial density at the site of infection was determined after imipenem treatment (30 and 60 mg/kg every 2 h for 24 h). The log(10) reduction in CFU/thigh was greatest for the wild-type isolate, intermediate for the two imipenem-susceptible VPKP isolates, and lowest for the imipenem-resistant VPKP isolate. Whilst in-vivo imipenem activity appeared reduced against in-vitro susceptible VIM-1 producers compared with a VIM-1-negative control, an increased drug dosage could moderate this reduction. PMID:17328735

6-Cyanopurine (6-CP) can be used as a color indicator for certain classes of nif (N2 fixation) mutations in Klebsiellapneumoniae. Under N2-fixing conditions, Nif+ colonies and most Nif- colonies are purple on media containing 6-CP. Twenty-two Nif- mutants with altered color on medium containing 6-CP were isolated. All white mutants contained mutations in the regulatory genes, nifAA-nifL. Mutants which were more darkly colored than the wild type had mutations distributed among six nif genes. Medium with 6-CP was used to isolate Nif- mutants with deletions internal to the nif genes, and 6-CP was used to identify strains depressed for nitrogenase synthesis in the presence of NH4+. PMID:361692

The in vitro activity of plazomicin was evaluated against 300 multidrug resistant (MDR) (carbapenemase and/or ESBL-producing) isolates from four hospitals in Athens, an area where carbapenemase-producing organisms are endemic. Most of the isolates were also resistant to the legacy aminoglycosides with the MIC50/MIC90 to tobramycin, amikacin and gentamicin being 32/>32, 32/>32 and 4/>8 μg/ml, respectively. ACHN-490 retained activity (MICs⩽4 μg/ml) against all isolates of Klebsiellapneumoniae, Escherichia coli, and Enterobacter spp. tested with MIC50 and MIC90 of 1 and 2 μg/ml, respectively, irrespective of their MDR phenotype and it represents a promising alternative for the treatment of the most problematic Gram-negative pathogens. PMID:23040681

1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiellapneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P21, with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 Å, β = 92.9°. The crystals probably contain two decamers in the asymmetric unit, with a V M value of 3.07 Å3 Da−1 and an estimated solvent content of 59%. Diffraction data were collected to 2.7 Å resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility. PMID:17329826

Between June 2009 and December 2013, 4105 patients were screened for carbapenem-resistant Klebsiellapneumoniae (CR-Kp) colonization in a tertiary care university hospital. The antimicrobial susceptibility and resistance determinants of 279 (6.8%) CR-Kp isolates from single patients were investigated. Additional analysis was performed to evaluate the characteristics and various risk factors for infection in patients with colonization. Of the 279 isolates, 270 harboured OXA-48-like enzymes, and a single isolate harboured IMP-type carbapenemase. A high proportion of isolates were susceptible to carbapenems - except ertapenem. All isolates were susceptible to amikacin and most (94%) were susceptible to colistin and fosfomycin. There was consistent high-level resistance for all isolates to temocillin, piperacillin-tazobactam, amoxicillin-clavulanate and ticarcillin-clavulanate. When colonized and infected patients were compared, only prior carbapenem administration (P = 0.003), was found to be significantly associated with patients with CR-Kp infection. PMID:27306118

The present study reports that Klebsiellapneumoniae (KP) killed the Caenorhabditis elegans as a consequence of an accumulation and proliferation of the pathogen inside the worms' intestine. The real-time PCR analysis of the genes responsible for vulval development (let-23) and egg laying (lin-29) in KP infected C. elegans confirmed the reproductive defects provoked by KP at the molecular level. In addition, the genetic analysis in N2 wild type, tol-1, sek-1 and pmk-1 mutants unveiled that KP attenuates the toll-dependent p38 mitogen-activated protein kinase (p38 MAPK) by chiefly inhibiting the production of antimicrobial factors such as nlp-29, lys-1 and C-type lectins. Conclusively, the surrendering of the host immune system appears to be attenuated by the toll-dependent p38 MAPK pathway regulation in C. elegans. PMID:25819035

The sequence type 11 Klebsiellapneumoniae strain Kpn-3002cz was confirmed to harbor two NDM-1-encoding plasmids, pB-3002cz and pS-3002cz. pB-3002cz (97,649 bp) displayed extensive sequence similarity with the blaNDM-1-carrying plasmid pKPX-1. pS-3002cz (73,581 bp) was found to consist of an IncR-related sequence (13,535 bp) and a mosaic region (60,046 bp). A 40,233-bp sequence of pS-3002cz was identical to the mosaic region of pB-3002cz, indicating the en bloc acquisition of the NDM-1-encoding region from one plasmid by the other. PMID:25421477

The sequence type 11 Klebsiellapneumoniae strain Kpn-3002cz was confirmed to harbor two NDM-1-encoding plasmids, pB-3002cz and pS-3002cz. pB-3002cz (97,649 bp) displayed extensive sequence similarity with the blaNDM-1-carrying plasmid pKPX-1. pS-3002cz (73,581 bp) was found to consist of an IncR-related sequence (13,535 bp) and a mosaic region (60,046 bp). A 40,233-bp sequence of pS-3002cz was identical to the mosaic region of pB-3002cz, indicating the en bloc acquisition of the NDM-1-encoding region from one plasmid by the other. PMID:25421477

The enzyme nitrogenase catalyzes the biological reduction of N/sub 2/ to NH/sub 3/. In Klebsiellapneumoniae a cluster of 17 genes in seven transcriptional units has been associated with nitrogen fixation. The nitrogenase enzyme from the nif V mutants is relatively ineffective at dinitrogen reduction, is more efficient than the wild-type enzyme at HCN reduction, and has its hydrogen evolution activity inhibited up to 80% by CO. This altered substrate specificity has been shown to be associated with the iron-molybdenum cofactor, FeMo-co, of the enzyme. X-ray absorption spectroscopy has been a valuable tool for probing the molybdenum environment of wild-type nitrogenase, and the authors report here similar studies on the Nif V/sup -/ enzyme.

The survival of B6D2F1 female mice exposed to lethal doses of fission neutron radiation is increased when trehalose dimycolate (TDM) preparations are given either 1 h after exposure or 1 day before exposure to radiation. TDM in an emulsion of squalene, Tween 80, and saline was the most effective formulation for increasing the 30-day survival of mice when given 1 day before (90%) or 1 h after (88%) exposure to radiation. An aqueous suspension of a synthetic analog of TDM was less effective at increasing 30-day survival (60%) when given 1 day prior to radiation exposure and not effective when given 1 h after radiation. Mice receiving a sublethal dose (3.5 Gy) of fission neutron radiation and either the TDM emulsion or synthetic TDM 1 h after irradiation were substantially more resistant to challenge with 10, 100, 1000, or 5000 times the LD50/30 dose of Klebsiellapneumoniae than untreated mice.

Background The emergence of carbapenem-resistant Klebsiellapneumoniae poses a serious problem to antibiotic management. We investigated the β-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey. Methods Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of β-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)-PCR analysis. Results All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like β-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M-1-like (62%) β-lactamases. Sequence analysis of blaTEM genes identified a blaTEM-166 with an amino acid change at position 53 (Arg53Gly) from blaTEM-1b, the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. Conclusions Combinations of β-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other β-lactamases, such as TEM, SHV, and CTX-M β-lactamases. PMID:26354347

We investigated the molecular epidemiology of extended spectrum β-lactamase (ESBL) producing Klebsiellapneumoniae isolates derived from the teaching hospitals of University of Pécs, Pécs, Hungary in the time period 2004-2008. Molecular typing, antimicrobial susceptibility testing, detection of common β-lactamase genes (bla(CTX-M), bla(TEM) and bla(SHV)) and virulence associated traits (hypermucoviscosity, magA, k2a, rmpA, siderophores, type 1 and 3 fimbria, biofilm formation, serum resistance) were performed for 102 isolates. The results showed the presence of three major ciprofloxacin resistant CTX-M-15 producing clones (ST15 n = 69, ST101 n = 10, and ST147 n = 9), of which ST15 was predominant and universally widespread. Considering distribution in time and place, ST101 and ST147 were detected at fewer inpatient units and within a narrower time frame, as compared to ST15. Beside major clones, eleven minor clones were identified, and were shown to harbour the following β-lactamase genes: six clones carried bla(CTX-M), four clones harboured bla(SHV-5) and one clone possessed both bla(CTX-M) and ESBL type bla(SHV). Among the SHV-5 producing K. pneumoniae clones a novel sequence type was found, namely ST1193, which harboured a unique infB allele. Different virulence factor content and peculiar antimicrobial susceptibility profile were characteristic for each clone. In contrast to major clone isolates, which showed high level resistance to ciprofloxacin, minor clone isolates displayed significantly lower MIC values for ciprofloxacin suggesting a role for fluoroquinolones in the dissemination of the major K. pneumoniae clones. This is the first description of the CTX-M-15 producing K. pneumoniae clone ST101 in Hungary. PMID:26551567

The minimal concentration of antibiotic required to inhibit the growth of different isolates of a given species with no acquired resistance mechanisms has a normal distribution. We have previously shown that the presence or absence of transmissible antibiotic resistance genes has excellent predictive power for phenotype. In this study, we analyzed the distribution of six β-lactam antibiotic susceptibility phenotypes associated with commonly acquired resistance genes in Enterobacteriaceae in Sydney, Australia. Escherichia coli (n = 200) and Klebsiellapneumoniae (n = 178) clinical isolates, with relevant transmissible resistance genes (blaTEM, n = 33; plasmid AmpC, n = 69; extended-spectrum β-lactamase [ESBL], n = 116; and carbapenemase, n = 100), were characterized. A group of 60 isolates with no phenotypic resistance to any antibiotics tested and carrying none of the important β-lactamase genes served as comparators. The MICs for all drug-bacterium combinations had a normal distribution, varying only in the presence of additional genes relevant to the phenotype or, for ertapenem resistance in K. pneumoniae, with a loss or change in the outer membrane porin protein OmpK36. We demonstrated mutations in ompK36 or absence of OmpK36 in all isolates in which reduced susceptibility to ertapenem (MIC, >1 mg/liter) was evident. Ertapenem nonsusceptibility in K. pneumoniae was most common in the context of an OmpK36 variant with an ESBL or AmpC gene. Surveillance strategies to define appropriate antimicrobial therapies should include genotype-phenotype relationships for all major transmissible resistance genes and the characterization of mutations in relevant porins in organisms, like K. pneumoniae. PMID:26912748

Studies on the prevalence and virulence genes of Klebsiella mastitis pathogens in a buffalo population are undocumented. Also, the association of rmpA kfu, uge, magA, Aerobactin, K1 and K2 virulent factors with K. pneumoniae buffalo, and cow mastitis is unreported. The virulence of K. pneumoniae was evaluated through both phenotypic and molecular assays. In vivo virulence was assessed by the Vero cell cytotoxicity, suckling mouse assay and mice lethality test. Antimicrobial susceptibility was tested by disk diffusion method. The 45 K. pneumoniae isolates from buffalo (n = 10/232) and cow (n = 35/293) milk were isolated (45/525; 8.6%) and screened via PCR for seven virulence genes encoding uridine diphosphate galactose 4 epimerase encoding gene responsible for capsule and smooth lipopolysaccharide synthesis (uge), siderophores (kfu and aerobactin), protectines or invasins (rmpA and magA), and the capsule and hypermucoviscosity (K1 and K2). The most common virulence genes were rmpA, kfu, uge, and magA (77.8% each). Aerobactin and K1 genes were found at medium rates of 66.7% each and K2 (55.6%). The Vero cell cytotoxicity and LD (50) in mice were found in 100% of isolates. A multidrug resistance pattern was observed for 40% of the antimicrobials. The distribution of virulence profiles indicate a role of rmpA, kfu, uge, magA, Aerobactin, and K1 and K2 in pathogenicity of K. pneumoniae in udder infections and invasiveness, and constitutes a threat for vulnerable animals, even more if they are in combination with antibiotic resistance. PMID:24915048

The emergence of multidrug-resistant (MDR) Klebsiellapneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Colr) is increasingly reported from clinical settings. The genetic mechanisms that lead to Colr in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Colr clinical isolates. Colr was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-l-arabinose (Ara4N) modification of lipid A was found in all Colr strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Colr mechanisms may be dependent on the genetic background. PMID:25385117

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiellapneumoniae are the leading causes of hospital-associated infections, but community-acquired cases are increasingly being reported. This study determined the prevalence of ESBL-producing E. coli and K. pneumoniae carriers, their bla genes and risk factors of 452 patients admitted to the emergency room (ER) of Ramathibodi Hospital, Mahidol University, Bangkok, Thailand between April and August 2011. Prevalence of ESBL-producing E. coli and K. pneumoniae from rectal swabs was 16.5% and 1.0%, respectively. Factors associated with ESBL-producing carriers were a previous history of hospital admission (p = 0.001) and visits to health care facilities (p = 0.002) during the previous 3 months. All ESBL-producing isolates were susceptible to imipenem, meropenem and ertapenem. The majority (78%) of ESBL-producing E. coli isolates showed very high resistance to cefotaxime and ceftriaxone (MIC50 and MIC90 > 256 µg/ml). ESBL-producing E. coli harbored chromosomal blaTEM (96%), blaCTX-M (70%) and blaSHV (1%), while 8%, 73% and 3%, respectively, were located on plasmid. The prevalence of these genes in ESBL-producing K. pneumoniae was 75%, 50% and 25%, respectively on chromosome; and 100%, 25% and 50%, respectively on plasmid. Nucleotide sequence analysis revealed that these bla genes were of the type blaTEM-1' blaTEM-116' blaCTX-M-15' blaCTX-M-161' blaSHV-12, blaSHV-28 and blaSHV-148. Detailed epidemiologic and clinical characteristics of ER patients with history of prior hospital visits should be carried out to identify the ESBL-producing organisms they have acquired in order to institute appropriate treatment for these patients as well as control measures against further dissemination of these life-threatening organisms. PMID:26513905

In order to get insights into plasmid evolution and the dissemination of multidrug resistance, we performed extensive comparative genomics analyses of the Klebsiellapneumoniae plasmid pKF3-94 and some of its related plasmids. pKF3-94 is one of three plasmids isolated from the K. pneumoniae strain KF3. Of the 144 putative genes it harbors, 69 can be functionally assigned to be involved in transfer conjugation, transfer leading, antimicrobial resistance, transposon function, and plasmid replication. Comparison of plasmid replicon sequence types revealed that pKF3-94 carries two replicons that are distinct from those carried on the two sibling K. pneumonia plasmids pKF3-70 and pKF3-140, thereby allowing pKF3-94 to coexist with these latter plasmids in the same host cell. Comparative genomics analyses further showed that pKF3-94 is more similar to plasmids pK1HV and pC15-k, which were isolated from different K. pneumonia strains, than to pKF3-70 and pKF3-140. Interestingly, pK1HV contains a unique 49 kb region rich in mobile genetic elements and drug resistance genes, while pKF3-94 and pC15-k share a 15 kb homology region partitioned into a region rich in drug resistance genes and one containing a replicon. It is conceivable, therefore, that pK1HV and pC15-k have both arisen from a common pKF3-94-like plasmid. The comparisons lend further support for the role horizontal gene transfer plays in genome evolution and in the dissemination of genetic elements including drug resistance genes. PMID:26347723

Objective To determine the rates of and risk factors for tigecycline non-susceptibility among carbapenem-resistant Klebsiellapneumoniae (CRKP) isolated from hospitalized patients. Design Multicenter prospective observational study Setting Acute care hospitals participating in the Consortium on Resistance against Carbapenems in Klebsiellapneumoniae (CRaCKle) Patients 287 patients who had CRKP isolated from clinical cultures during hospitalization Methods Within the study period of 12/24/2011 – 10/1/2013, the first hospitalization of each patient with CRKP was included during which tigecycline susceptibility for the CRKP isolate was determined. Clinical data was entered into a centralized database, including data on pre-hospital origin. Breakpoints established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to interpret tigecycline susceptibility testing. Results Of 287 patients included, 155 (54%) had tigecycline-susceptible CRKP, whereas 81 (28%) of index isolates were tigecycline-intermediate, and 51 (18%) were tigecycline-resistant. In multivariable modeling, admission from a skilled nursing facility (OR 2.51, 95% CI 1.51–4.21, p=0.0004), positive culture within 2 days of admission (OR 1.82, 95% CI 1.06–3.15, p=0.03), and receipt of tigecycline within 14 days (OR 4.38, 95% CI 1.37–17.01, p=0.02) were found to be independent risk factors for tigecycline non-susceptibility. Conclusions In hospitalized patients with CRKP, tigecycline non-susceptibility was more frequently seen in admissions from skilled nursing facilities and occurred earlier during hospitalization. Skilled nursing facilities are an important target for interventions to decrease antibacterial resistance to antibiotics of last resort for treatment of CRKP. PMID:25990806

Klebsiellapneumoniae is an opportunistic pathogen that causes several kinds of infections, including pneumonia, bacteremia, urinary tract infection and community-acquired pyogenic liver abscess (PLA). Adhesion is the critical first step in the infection process. Our previous work demonstrated that the transcellular translocation is exploited by K. pneumoniae strains to migrate from the gut flora into other tissues, resulting in systemic infections. However, the initial stages of K. pneumoniae infection remain unclear. In this study, we demonstrated that a K. pneumoniae strain deleted for yfgL (bamB) exhibited reduced adherence to and invasion of host cells; changed biogenesis of major β-barrel outer membrane proteins; decreased transcriptional expression of type-1 fimbriae; and increased susceptibility to vancomycin and erythromycin. The yfgL deletion mutant also had reduced ability to against neutrophil phagocytosis; exhibited decreased induction of host IL-6 production; and was profoundly attenuated for virulence in a K. pneumoniae model of bacteremia. Thus, the K. pneumoniae YfgL lipoprotein mediates in outer membrane proteins biogenesis and is crucial for anti-phagocytosis and survival in vivo. These data provide a new insight for K. pneumoniae attachment and such knowledge could facilitate preventive therapies or alternative therapies against K. pneumoniae. PMID:27029012

We characterized carbapenem resistance mechanisms among 12 Klebsiellapneumoniae carbapenemase (KPC)-producing K. pneumoniae (referred to here as KPC K. pneumoniae) clinical isolates and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem, and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to the ST258 clonal group and harbored blaKPC-2, blaSHV-12, and blaTEM-1. As determined by time-kill assays, doripenem (8 μg/ml) and ertapenem (2 μg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 μg/ml) exerted some activity (range, 0.39 to 2.5 log10) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem, and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12), and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other (R2 = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than those with low expression (100% [8/8] versus 0% [0/4]; P = 0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem (P ≤ 0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC K. pneumoniae, including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC K. pneumoniae isolates may be a practical tool for identifying effective combination regimens. PMID:23459476

We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiellapneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST. PMID:26014928

We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiellapneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST. PMID:26014928

S-thanatin (Ts) was a short antimicrobial peptide with selective antibacterial activity. In this study, we aimed to design a drug carrier with specific bacterial targeting potential. The positively charged Ts was modified onto the liposome surface by linking Ts to the constituent lipids via a PEG linker. The benefits of this design were evaluated by preparing a series of liposomes and comparing their biological effects in vitro and in vivo. The particle size and Zeta potential of the constructed liposomes were measured with a Zetasizer Nano ZS system and a confocal laser scanning microscope. The in vitro drug delivery potential was evaluated by measuring the cellular uptake of encapsulated levofloxacin using HPLC. Ts-linked liposome or its conjugates with quantum dots favored bacterial cells, and increased the bacterial uptake of levofloxacin. In antimicrobial assays, the Ts and levofloxacin combination showed a synergistic effect, and Ts-LPs-LEV exhibited excellent activity against the quality control stain Klebsiellapneumoniae ATCC 700603 and restored the susceptibility of multidrug-resistant K. pneumoniae clinical isolates to levofloxacin in vitro. Furthermore, Ts-LPs-LEV markedly reduced the lethality rate of the septic shock and resulted in rapid bacterial clearance in mouse models receiving clinical multidrug resistant (MDR) isolates. These results suggest that the Ts-functionalized liposome may be a promising antibiotic delivery system for clinical infectious disorders caused by MDR bacteria, in particular the sepsis related diseases. PMID:26578959

Objective: There is an increased prevalence of extended-spectrum beta-lactamase producing Klebsiellapneumoniae (ESBL-KP) worldwide including India, which is a major concern for the clinicians, especially in intensive care units and pediatric patients. This study aims to determine the prevalence of ESBL-KP and antimicrobial sensitivity profile to plan a proper hospital infection control program to prevent the spread of resistant strains. Methods: KP isolates obtained from various clinical samples were evaluated to detect the production of ESBL by phenotypic methods. Antimicrobial susceptibility profile was also determined of all the isolates. Findings: Of 223 nonduplicate isolates of K. pneumoniae, 114 (51.1%) were ESBL producer and antimicrobial susceptibility profile showed the isolates were uniformly sensitive to imipenem and highly susceptible to beta-lactamase inhibitor combination drugs (67–81%) and aminoglycosides (62–76%), but less susceptible to third generation cephalosporins (14–24%) and non-β-lactam antibiotics such as nitrofurantoin (57%), fluoroquinolones (29–57%), piperacillin (19–23%), and aztreonam (15–24%). Conclusion: This study found that beta-lactamase inhibitor combinations are effective in treatment of such infections due to ESBL-KP thus these drugs should be a part of the empirical therapy and carbapenems should be used when the antimicrobial susceptibility tests report resistance against inhibitors combinations. PMID:26312255

To date, few studies have focused on reducing the toxic by-product acetate during 1,3-propanediol production by Klebsiellapneumoniae. In this study, the effects of deleting the poxB, pta, and ackA genes, which are involved in the two main acetate synthesis pathways, on cell growth and 1,3-propanediol production were investigated. Although acetate synthesis via pyruvate oxidase (PoxB, encoded by poxB) generally seems unnecessary and wasteful, PoxB was shown to play an important role in K. pneumoniae. Deletion of poxB severely inhibited cell growth, and the poxB mutant exhibited an anomalously high accumulation of acetate in aerobic cultures and failed to produce an endogenous supply of carbon dioxide (CO2) in anaerobic cultures. It is interesting that both the aerobic and anaerobic growth defects of the poxB mutant were corrected by further deleting pta and ackA, which blocked the other main acetate synthesis pathway. The poxB-pta-ackA mutant excreted less acetate and showed an excellent ability to produce 1,3-propandiol. The final 1,3-propanediol yield and concentration in a 2-L fed-batch fermentation reached 0.66 (mol/mol) and 76.8 g/L, respectively, which were 16 and 15 % greater, respectively, than those of the parent strain. PMID:26695159

In this study susceptibility to different antimicrobial peptides was investigated on colistin-susceptible and colistin-resistant identical pulsotype strains of KPC-2 producing Klebsiellapneumoniae ST258 as well as colistin-susceptible and colistin-resistant Enterobacter asburiae strains isolated from clinical samples. In our test, bacteria were exposed to 50 mg/ml lactoferrin, lysozyme and protamine - cationic antimicrobial peptides belonging to innate immune system and having structural similarity to polymyxins - in separate reactions. After 18 hours incubation of colonies were counted. 40% of colistin-resistant K. pneumoniae strains and 97% of colistin-susceptible counterpart strains were lysed by protamine whereas 87% and 100% colony forming unit decrease by lysozyme was seen, respectively. In the case of colistin-resistant E. asburiae strains 1 log10 cell count increase were observed after treatment with lysozyme and 1.56 log10 after lactoferrin exposure compared to the initial number whereas the colistin-susceptible showed no relevant cell count increase. Our findings suggest that acquired colistin-resistance in Enterobacteriaceae is associated with tolerance against antimicrobial peptides. PMID:26689883

This study describes a syndrome of neonatal septicemia and meningitis in New Zealand sea lions, caused by a strain of Klebsiellapneumoniae that is phenotypically similar to strains causing environmentally-acquired septicemia and neuro-invasive disease in humans. Between late 2006 and early 2010, 123 pups from the Enderby Island breeding colony died of K. pneumoniae infection, with lesions including fibrinous to fibrinosuppurative meningitis, subdural hemorrhage, septic arthritis, herniation and hemorrhage of the cerebellar vermis, lymphadenitis and cellulitis. This infection was responsible for 58% of observed pup mortality over this time period, with most deaths occurring in the latter part of the breeding season (mid February onwards). The results of this study suggest that the pattern of this disease has changed since it was first described in 2002, when most deaths occurred early in the season (early to mid-January), and that it is an important and consistent cause of pup mortality in this population. In addition, a similar disease syndrome and bacterial strain was diagnosed in a single pup in a fragile recolonizing New Zealand sea lion population on mainland New Zealand, and the potential effect on this population is unknown but could have a negative impact on recolonisation at this site. PMID:25682024

The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacter freundii and Klebsiellapneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia. PMID:8017933

Multidrug-resistant Klebsiellapneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258. PMID:26196384

Butanol isomers are important bulk chemicals and promising fuel substitutes. The inevitable toxicity of n-butanol and isobutanol to microbial cells hinders their final titers. In this study, we attempt to engineer Klebsiellapneumoniae for the de novo production of 2-butanol, another butanol isomer which shows lower toxicity than n-butanol and isobutanol. 2-Butanol synthesis was realized by the extension of the native meso-2,3-butanediol synthesis pathway with the introduction of diol dehydratase and secondary alcohol dehydrogenase. By the screening of different secondary alcohol dehydrogenases and diol dehydratases, 320mg/L of 2-butanol was produced by the best engineered K. pneumoniae. The production was increased to 720mg/L by knocking out the ldhA gene and appropriate addition of coenzyme B12. Further improvement of 2-butanol to 1030mg/L was achieved by protein engineering of diol dehydratase. This work lays the basis for the metabolic engineering of microorganism for the production of 2-butanol as potential biofuel. PMID:26342337

In Finland, occurrence of Klebsiellapneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has previously been sporadic and related to travel. We describe the first outbreak of colonisation with KPC-KP strain ST512; it affected nine patients in a 137-bed primary care hospital. The index case was detected by chance when a non-prescribed urine culture was taken from an asymptomatic patient with suprapubic urinary catheter in June 2013. Thereafter, all patients on the 38-bed ward were screened until two screening rounds were negative and extensive control measures were performed. Eight additional KPC-KP-carriers were found, and the highest prevalence of carriers on the ward was nine of 38. All other patients hospitalised on the outbreak ward between 1 May and 10 June and 101 former roommates of KPC-KP carriers since January had negative screening results. Two screening rounds on the hospital's other wards were negative. No link to travel abroad was detected. Compared with non-carriers, but without statistical significance, KPC-KP carriers were older (83 vs 76 years) and had more often received antimicrobial treatment within the three months before screening (9/9 vs 90/133). No clinical infections occurred during the six-month follow-up. Early detection, prompt control measures and repetitive screening were crucial in controlling the outbreak. PMID:26159309

1,3-Propanediol dehydrogenase from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.7 Å resolution. 1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiellapneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 Å, β = 92.9°. The crystals probably contain two decamers in the asymmetric unit, with a V{sub M} value of 3.07 Å{sup 3} Da{sup −1} and an estimated solvent content of 59%. Diffraction data were collected to 2.7 Å resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility.

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiellapneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively. 22 references.

Klebsiellapneumoniae, a worldwide cause of nosocomial infections, is one of the most common causes of death in newborns in nurseries. In this study, we investigated the role of interleukin-1 (IL-1) in an experimental animal model of neonatal sepsis, using a natural antagonist of IL-1 receptors, the IL-1 receptor antagonist (IL-1Ra), to block IL-1's effects in neonatal Klebsiella sepsis in the absence of antibiotic treatment. Newborn Wistar-Kyoto rats were injected intraperitoneally with a single dose (10 mg/kg) of either IL-1Ra (n = 43) or human serum albumin as a control (n = 40). At the same time, a 50% lethal dose of K. pneumoniae was injected subcutaneously. No antibiotics were given at any time. After 10 days, survival was 60% for the albumin group and 80% for the IL-1Ra group (P < 0.01). IL-1Ra treatment also afforded protection when the dose of bacteria was increased sixfold (P < 0.01). There were two episodes of leukopenia in the control group, which were suppressed by IL-1Ra (P < 0.01 and P < 0.001). IL-1 and IL-6 levels were lower in the IL-1Ra-treated group (P < 0.05 and P < 0.001, respectively). No differences between the two groups were observed in the number of bacteria in cultures of the blood, lungs, liver, or spleen. When IL-1Ra (10 mg/kg) was given both at time zero and 24 h after bacterial challenge, lethality was significantly increased (P < 0.01). Single doses of IL-1Ra of from 20 to 40 mg/kg progressively increased lethality compared with controls (P < 0.01) in both Wistar-Kyoto and Sprague-Dawley strain rats. In the same model, low doses of IL-1 itself (0.4 ng per rat), given 24 h prior to bacterial challenge, afforded protection (P < 0.001). These studies suggest that, in the absence of antibiotics, partial blockade of IL-1 receptors improves survival, whereas a longer or greater blockade increases lethality in newborn rats infected with K. pneumoniae. PMID:8432613

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiellapneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost. PMID:26282408

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiellapneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches. PMID:25751658

Introduction Lemierre's syndrome was originally documented to be caused by Fusobacterium necrophorum. It is a very rare condition with a prevalence of one to 14.4 instances per million. Its presentation is varied, not only in composition but also in the infecting organism. Treatment with anticoagulants has been controversial and applied only on a case-by-case basis. Case presentation A 63-year-old Saudi man who had had uncontrolled diabetes mellitus for 47 years presented to our facility with a five-day history of swelling on the right side of his neck and fever. The swelling progressively increased in size and was associated with pain, dysphagia, odynophagia, change of voice ('hot potato voice'), and reduced appetite. Abscess content culture and sensitivity testing revealed Klebsiellapneumoniae. However, blood culture results were repeatedly negative. The abscess was incised and drained without any complication. Our patient was treated with clindamycin and cefuroxime. Warfarin was also administered concurrently for six weeks, for an isolated internal jugular vein thrombosis (IJV), with complete resolution of the thrombus. Normoglycemia was achieved and our patient was discharged after complete wound healing and the return of his biochemical parameters to normal. Conclusions Only two cases of Lemierre's syndrome in patients with diabetes due to K. pneumoniae have been reported previously. A review of the literature suggested that an association exists between deep neck infections due to K. pneumoniae and diabetes mellitus. The reasons for this association are still not clear. This poses a question as to whether diabetes mellitus specifically predisposes these patients to infection with this organism. It is suggested that clinicians should consider infectious agents other than F. necrophorum in the causation of Lemierre's syndrome, especially in patients with diabetes. PMID:22472458

With the increase in extended spectrum β-lactamase (ESBL)-producing bacteria in the community, cases are often seen in which treatment of infectious diseases with oral antimicrobial agents is difficult. Therefore, we measured the antimicrobial activities of 14 currently available oral antimicrobial agents against ESBL-producing Escherichia coli and Klebsiellapneumoniae. Based on the standard of the Clinical and Laboratory Standards Institute (CLSI), E. coli showed high susceptibility rates of 99.4% to faropenem (FRPM). In terms of fluoroquinolones, the susceptibility rate of E. coli to levofloxacin (LVFX) was low at 32.2%, whereas it showed a good susceptibility rate of 93.1% to sitafloxacin (STFX). With respect to other antimicrobial agents, susceptibility rates to fosfomycin (FOM) and colistin (CL) were more than 90% each, whereas rates of the two antimicrobial agents expected as therapeutic agents, minocycline (MINO) and sulfamethoxazole-trimethoprim (ST), were low at 62.4% and 44.3%, respectively. Based on the CLSI standard, K. pneumoniae showed high susceptibility rates to ceftibuten (CETB) (91.89%), LVFX (86.49%), and STFX (94.6%), indicating that K. pneumoniae showed higher rates than those of E. coli, particularly to fluoroquinolones. Comparison of susceptibility rates according to E. coli genotype showed that many antimicrobial agents existed to which the CTX-M-9 group showed high susceptibility rates. However, there were many agents to which the CTX-M-1 group showed low susceptibility rates, particularly to CETB (51.1%) and LVFX (17.0%). Although there was no significant difference by genotype between FRPM, STFX, and FOM, a significant difference was observed between LVFX, MINO, and ST. Antibiotic-resistant bacteria with highly pathogenic strains have spread in the community, appropriate use of oral antimicrobial agents is required. PMID:24462425

Oxygen caused a reversible inhibition (switch-off) of nitrogenase activity in whole cells of four strains of diazotrophs, the facultative anaerobe Klebsiellapneumoniae and three strains of photosynthetic bacteria (Rhodopseudomonas sphaeroides f. sp. denitrificans and Rhodopseudomonas capsulata strians AD2 and BK5). In K. pneumoniae 50% inhibition of acetylene reduction was attained at an O/sub 2/ concentration of 0.37 ..mu..M. Cyanide (90 ..mu..M), which did not affect acetylene reduction but inhibited whole-cell respiration by 60 to 70%, shifted the O/sub 2/ concentration that caused 50% inhibition of nitrogenase activity to 2.9 ..mu..M. A mutant strain of K. pneumoniae, strain AH11, has a respiration rate that is 65 to 75% higher than that of the wild type, but is nitrogenase activity is similar to wild-type activity. Acetylene reduction by whole cells of this mutant was inhibited 50% by 0.20 ..mu..M O/sub 2/. Inhibition by CN/sup -/ of 40 to 50% of the O/sub 2/ uptake in the mutant shifted the O/sub 2/ concentration that caused 50% inhibition of nitrogenase to 1.58 ..mu..M. Thus, when the respiration rates were lower, higher oxygen concentrations were required to inhibit nitrogenase. Reversible inhibition of nitrogenase activity in vivo was caused under anaerobic conditions by other electron acceptors. Addition of 2 mM sulfite to cell suspensions of R. capsulata B10 and R. sphaeroides inhibited nitrogenase activity. Nitrite also inhibited acetylene reduction in whole cells of the photodenitrifier R. sphaeroides but not in R. capsulata B10, which is not capable of enzymatic reduction of NO/sub 2//sup -/. Lower concentrations of NO/sub 2//sup -/ were required to inhibit the activity in NO/sub 3//sup -/-grown cells, which have higher activities of nitrite reductase.

Objectives Combination therapy is an important option in the fight against Gram-negative ‘superbugs’. This study systematically investigated bacterial killing and the emergence of polymyxin resistance with polymyxin B and chloramphenicol combinations used against New Delhi metallo-β-lactamase (NDM)-producing MDR Klebsiellapneumoniae. Methods Four NDM-producing K. pneumoniae strains were employed. The presence of genes conferring resistance to chloramphenicol was examined by PCR. Time–kill studies (inocula ∼106 cfu/mL) were conducted using various clinically achievable concentrations of each antibiotic (range: polymyxin B, 0.5–2 mg/L; chloramphenicol, 4–32 mg/L), with real-time population analysis profiles documented at baseline and 24 h. The microbiological response was examined using the log change method and pharmacodynamic modelling in conjunction with scanning electron microscopy (SEM). Results Multiple genes coding for efflux pumps involved in chloramphenicol resistance were present in all strains. Polymyxin B monotherapy at all concentrations produced rapid bacterial killing followed by rapid regrowth with the emergence of polymyxin resistance; chloramphenicol monotherapy was largely ineffective. Combination therapy significantly delayed regrowth, with synergy observed in 25 out of 28 cases at both 6 and 24 h; at 24 h, no viable bacterial cells were detected in 15 out of 28 cases with various combinations across all strains. No polymyxin-resistant bacteria were detected with combination therapy. These results were supported by pharmacodynamic modelling. SEM revealed significant morphological changes following treatment with polymyxin B both alone and in combination. Conclusions The combination of polymyxin B and chloramphenicol used against NDM-producing MDR K. pneumoniae substantially enhanced bacterial killing and suppressed the emergence of polymyxin resistance. PMID:26023209

In the present study, we describe an outbreak caused by extended-spectrum beta-lactamase (ESBL) producing Klebsiellapneumoniae in the neonatal intensive care unit (NICU) of a tertiary-care hospital. Nosocomial blood-stream infections were detected in three patients hospitalized in NICU. Two of the cases were transferred to NICU due to premature birth and the other due to the presence of cleft palate and retrognathia. K.pneumoniae was isolated on the 5th day of hospitalization of the first patient from umbilical swab and blood culture; on the 15th day of hospitalization of the second patient from blood culture and on the 7th day of hospitalization of the third patient from blood culture. The isolates were identified by automated API Rapid ID 32 Staph (BioMerieux, France) system in addition to conventional laboratory methods. Antibiotic susceptibilities of the isolates were determined by using Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) criteria. The same antibiotic susceptibility patterns were detected in all isolates. Active surveillance cultures included environmental sampling from surfaces of NICU, laryngoscopes, ventilators and connections of ventilators, stethoscopes, nebulizers, aspiration tubings, disinfectant solutions, couveuse and couveuse distilled water. Two ESBL producing K.pneumoniae strains, presenting the same antibiotic susceptibility pattern with the clinical strains, were isolated from one couveuse distilled water sample and one aspiration tubing. All of the K.pneumoniae isolates were resistant to amoxycillin-clavulonic acid, cefazolin, cefepime, ceftriaxone, ceftazidime, cefuroxime, aztreonam and trimetoprim-sulphametoxazole and susceptible to cefoxitin, imipenem, meropenem, gentamicin, tobramycin, amikacin, netilmisin, tetracycline, ciprofloxacin and chloramphenicol. Arbitrarily primed polymerase chain reaction (AP-PCR) analysis done with M13 primer revealed the same genotype for the patient

Municipal wastewater treatment facilities are considered to be “hotspots” for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7–9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3

Municipal wastewater treatment facilities are considered to be "hotspots" for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7-9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like

The aim of this study was to investigate the prevalence and types of plasmid-mediated AmpC (pAmpC) beta-lactamase enzymes in Escherichia coli and Klebsiellapneumoniae strains isolated from blood cultures of hospitalized patients in Dokuz Eylul University Hospital between 2007 and 2012. A total of 261 isolates which consisted of 184 E.coli (70.5%) and 77 K.pneumoniae (29.5%) were included in the study. All isolates were resistant to cefotaxime and/or ceftazidime but susceptible to imipenem. Cefoxitin resistance was investigated as an indicator of AmpC type enzymes. A total of 57 (21.8%) isolates which were cefoxitin-resistant (32 E.coli, 25 K.pneumoniae), were screened for pampC genes by a multiplex polymerase chain reaction (PCR) assay. Additionally, 10 of each cefoxitin susceptible isolates per year were chosen randomly and screened by the same PCR assay to detect the presence of ACC enzymes, which can not hydrolyze cefoxitin. Positive PCR results were confirmed by sequence analysis. Plasmid analysis and macrorestriction analysis were performed for pampC-positive isolates. The presence of pAmpC enzymes has been shown in 9.4% (3/32) of cefoxitin-resistant E.coli, and 8% (2/25) of cefoxitin-resistant K.pneumoniae strains. It was noted that there were no strains producing this enzyme isolated in 2007 and 2008, however the prevalence of pAmpC was detected as 1.6% in 2009 (one ACT-1 producing K.pneumoniae), increasing to 4.8% in 2011 (one ACT-1 producing K.pneumoniae) and 6.4% in 2012 (three CMY-2 producing E.coli). These enzymes were found to be carried on 81 kb size plasmids in K.pneumoniae isolates and on a 9 kb size plasmid in E.coli isolates. Macrorestriction analysis indicated that two of the three CMY-2 producing E.coli had the same PFGE (Pulsed-field gel electrophoresis) pattern. If these two strains are considered as identical, it can be concluded that the prevalence of pAmpC was low in the strains isolated between 2007-2012 (4/261; 1.5%) in our institution

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiellapneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity. PMID:11443073

A “high risk” clone of carbapenem-resistant Klebsiellapneumoniae (CRKP) identified by multilocus sequence typing (MLST) as sequence type (ST) 258 has disseminated worldwide. As the molecular epidemiology of the CRE pandemic continues to evolve, the clinical impact of non-ST258 strains is less well defined. We conducted an epidemiological investigation of CRKP based on strains MLST. Among 68 CRKP patients, 61 were ST258 and 7 belonged to non-ST258. Klebsiellapneumoniae ST258 strains were significantly associated with blaKPC production and with resistance to an increased number of antimicrobials. Clinical outcomes were not different. Based on this analysis, one cannot rely solely on the presence of blaKPC in order to diagnose CRKP. PMID:26885543

A "high risk" clone of carbapenem-resistant Klebsiellapneumoniae (CRKP) identified by multilocus sequence typing (MLST) as sequence type (ST) 258 has disseminated worldwide. As the molecular epidemiology of the CRE pandemic continues to evolve, the clinical impact of non-ST258 strains is less well defined. We conducted an epidemiological investigation of CRKP based on strains MLST. Among 68 CRKP patients, 61 were ST258 and 7 belonged to non-ST258. Klebsiellapneumoniae ST258 strains were significantly associated with bla KPC production and with resistance to an increased number of antimicrobials. Clinical outcomes were not different. Based on this analysis, one cannot rely solely on the presence of bla KPC in order to diagnose CRKP. PMID:26885543

17-year-old man had been involved in a traffic accident. He underwent a bilateral craniotomy with artificial dura mater to remove bilateral acute subdural hematomas. Seven months later, a right cranioplasty was performed using frozen auto-bone, and he developed extended-spectrum β-lactamase (ESBL)-producing Klebsiellapneumoniae meningitis and an epidural abscess. Since his general status was poor, we could not remove the foreign body (artificial dura mater). He was successfully treated with meropenem and chronic suppression with oral trimethoprim-sulfamethoxazole. By describing this case and the results of a review of the pertinent literature, we discuss the importance of ESBL-producing Klebsiellapneumoniae meningitis in posttraumatic/postoperative patients. PMID:22989843

In the facultative anaerobe Klebsiellapneumoniae 17 nitrogen fixation-specific genes (nif genes) have been identified. Homologs to 12 of these genes have now been isolated from the aerobic diazotroph Azotobacter vinelandii. Comparative studies have indicated that these diverse microorganisms share striking similarities in the genetic organization of their nif genes and in the primary structure of their individual nif gene products. In this study the complete nucleotide sequence of the nifUSV gene clusters from both K. pneumoniae and A. vinelandii were determined. These genes are identically organized on their respective genomes, and the individual genes and their products exhibit a high degree of interspecies sequence homology. PMID:3040672

Delafloxacin is a broad-spectrum anionic fluoroquinolone under development for the treatment of bacterial pneumonia. The goal of the study was to determine the pharmacokinetic/pharmacodynamic (PK/PD) targets in the murine lung infection model for Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiellapneumoniae Four isolates of each species were utilized for in vivo studies: for S. aureus, one methicillin-susceptible and three methicillin-resistant isolates; S. pneumoniae, two penicillin-susceptible and two penicillin-resistant isolates; K. pneumoniae, one wild-type and three extended-spectrum beta-lactamase-producing isolates. MICs were determined using CLSI methods. A neutropenic murine lung infection model was utilized for all treatment studies, and drug dosing was by the subcutaneous route. Single-dose plasma pharmacokinetics was determined in the mouse model after administration of 2.5, 10, 40, and 160 mg/kg. For in vivo studies, 4-fold-increasing doses of delafloxacin (range, 0.03 to 160 mg/kg) were administered every 6 h (q6h) to infected mice. Treatment outcome was measured by determining organism burden in the lung (CFU counts) at the end of each experiment (24 h). The Hill equation for maximum effect (Emax) was used to model the dose-response data. The magnitude of the PK/PD index, the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC), associated with net stasis and 1-log kill endpoints was determined in the lung model for all isolates. MICs ranged from 0.004 to 1 mg/liter. Single-dose PK parameter ranges include the following: for maximum concentration of drug in serum (Cmax), 2 to 70.7 mg/liter; AUC from 0 h to infinity (AUC0-∞), 2.8 to 152 mg · h/liter; half-life (t1/2), 0.7 to 1 h. At the start of therapy mice had 6.3 ± 0.09 log10 CFU/lung. In control mice the organism burden increased 2.1 ± 0.44 log10 CFU/lung over the study period. There was a relatively steep dose-response relationship

The entire plasmid content of a multidrug-resistant, CTX-M-15-producing Klebsiellapneumoniae ST416 clone was investigated. Two FII(K) plasmids, pKDO1 (127 kb) and pKPN-CZ (207 kb), were identified and found to carry a formidable set of genes conferring resistance to toxic compounds, metals, and antimicrobial drugs and exhibiting novel features putatively associated with adaptation and fitness of the bacterium in the human host. PMID:23229477

The entire plasmid content of a multidrug-resistant, CTX-M-15-producing Klebsiellapneumoniae ST416 clone was investigated. Two FIIK plasmids, pKDO1 (127 kb) and pKPN-CZ (207 kb), were identified and found to carry a formidable set of genes conferring resistance to toxic compounds, metals, and antimicrobial drugs and exhibiting novel features putatively associated with adaptation and fitness of the bacterium in the human host. PMID:23229477

The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

The efficacies of tigecycline and ceftazidime against fatal pneumonia in rats caused by an extended-spectrum β-lactamase (ESBL)-positive Klebsiellapneumoniae strain or its wild-type (WT) progenitor were compared. Ceftazidime at 12.5 or 50 mg/kg of body weight twice daily (b.i.d.) was effective (50% or 100% rat survival) in pneumonia caused by the WT isolate but unsuccessful (100% rat mortality) in pneumonia caused by the ESBL-positive variant. In contrast, tigecycline at 6.25, 12.5, or 25 mg/kg b.i.d. showed dosage-dependent efficacy up to 100% rat survival irrespective of the ESBL character of the infecting organism. PMID:23129049

Klebsiellapneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted. PMID:26872343

The aim of this work was to determine the genetic environment and transferability of blaKPC as well as the pulsotypes of KPC-producing Klebsiellapneumoniae strains isolated from clinical samples in Chilean hospitals. Seventeen strains, principally isolated in Santiago (the capital of Chile) during the years 2012 and 2013, were included. The genetic environment of blaKPC was elucidated by PCR mapping and sequencing. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Curing and conjugation experiments were performed with six strains of different sequence types (STs) and pulsotypes. Thirteen pulsotypes and six STs, mainly belonging to clonal complex 258, were found. In addition, seven strains belonged to a new ST assigned ST1161. The blaKPC sequence indicated that 16 strains had the KPC-2 variant; in only one strain (UC331) an amino acid change (R6P) was detected, corresponding to a new KPC variant designated KPC-24. Molecular characterisation of the blaKPC genetic environment revealed two distinct platforms, namely variant 1a and the Tn4401a isoform, with the first being the most common (11/17 strains). Mating experiments failed to produce transconjugants; however, loss of blaKPC was achieved by plasmid curing in all assayed strains. In conclusion, in Chilean strains of K. pneumoniae, blaKPC is primarily found associated with the variant 1a and is located in non-transferable plasmids. In addition, this study highlights the description of the new ST1161 and the new KPC-24 variant. PMID:27436389

Chlorite dismutases (Clds) convert chlorite to O2 and Cl(-), stabilizing heme in the presence of strong oxidants and forming the O═O bond with high efficiency. The enzyme from the pathogen Klebsiellapneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite. PMID:25437493

The Klebsiellapneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1. Images PMID:2186362

Tigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. In Enterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated in Enterobacteriaceae and Acinetobacter isolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiellapneumoniae isolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 μg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression of ramA, ramR, oqxA, acrB, marA, or rarA genes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5 insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here named kpgABC, previously unknown to be involved in resistance. Introduction of the kpgABC genes in a non-kpgABC background increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function for kpgABC and identify an insertion element whose presence correlated with the in vivo development of tigecycline nonsusceptibility in K. pneumoniae. PMID:25092708

This study investigated the transfer frequency of the extended-spectrum β-lactamase-encoding gene (blaSHV18) among Klebsiellapneumoniae in tryptic soy broth (TSB), pasteurized milk, unpasteurized milk, alfalfa sprouts and chopped lettuce at defined temperatures. All transconjugants were characterized phenotypically and genotypically. KP04(ΔKM) and KP08(ΔKM) isolated from seed sprouts and KP342 were used as recipients in mating experiments with K. pneumoniae ATCC 700603 serving as the donor. In mating experiments, no transconjugants were detected at 4 °C in liquid media or chopped lettuce, but detected in all media tested at 15 °C, 24 °C, and 37 °C. At 24 °C, the transfer of blaSHV18 gene occurred more frequently in alfalfa sprouts (5.15E-04 transconjugants per recipient) and chopped lettuce (3.85E-05) than liquid media (1.08E-05). On chopped lettuce, transconjugants were not detected at day 1 post-mating at 15 °C, but observed on day 2 (1.43E-05). Transconjugants carried the blaSHV18 gene transferred from the donor and the virulence gene harbored by recipient. More importantly, a class 1 integrase gene and resistance to tetracycline, trimethoprim/sulfamethoxazole were co-transferred during mating. These quantitative results suggest that fresh produce exposed to temperature abuse may serve as a competent vehicle for the spread of gene encoding for antibiotic resistance, having a potential negative impact on human health. PMID:27554144

Chlorite dismutases (Clds) convert chlorite to O2 and Cl–, stabilizing heme in the presence of strong oxidants and forming the O=O bond with high efficiency. The enzyme from the pathogen Klebsiellapneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite. PMID:25437493

Klebsiellapneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3−-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5′-CTTAAT-3′ and 5′-CCTAAG-3′, in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC–DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF–DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation. PMID:24371275

Dual oxidase 2 (DUOX2) plays a major role in host defense in intestinal and airway epithelial cells through the reactive oxygen species (ROS) pathway. Klebsiellapneumoniae is a uropathogen that causes urinary tract infections. It is not known whether DUOX2 plays a role in host defense in bladder cancer epithelial cells. It is also not known whether Klebsiellapneumoniae invades T24 human bladder carcinoma cells and whether DUOX2 plays a role in eliminating the Klebsiellapneumoniae strain K5 through the ROS pathway in T24 cells. Thus, in the present study, we aimed to investigate the infectious capability of the Klebsiellapneumoniae K5 strain and the immunity-promoting capability of DUOX2 in T24 cells. We quantified the number of viable intracellular bacteria using the plate count method. DUOX2 expression was evaluated by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR) following treatment with or without multiple cytokines, phorbol 12-myristate 13-acetate (PMA), muramyl dipeptide (MDP), N-acetylmuramyl-D-alanyl-D-isoglutamine (MDP-DD), H2O2 inhibitor, catalase (CAT), the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase inhibitor, diphenyleneiodonium (DPI), or siRNA targeting DUOX2 (siDUOX2). The levels of ROS in the T24 cells infected with the K5 strain were examined following treatment with DPI, CAT or siDUOX2. Our results revealed that DUOX2 expression increased and the number of viable intracellular bacteria decreased in the T24 cells following infection with the K4 bacteria. Treatment with the cytokines and MDP and PMA also induced DUOX2 expression and decreased the number of viable intracellular bacteria. The levels of ROS also increased following treatment with the cytokines and MDP and PMA. However, when the cells were treated with the inhibitors (DPI or CAT), these effects were all reversed. Our data demonstrated that DUOX2 played an important role in innate immunity against bacterial cytoinvasion through the

In this prospective study, consecutive isolates of Klebsiellapneumoniae were tested for different mechanisms of carbapenem resistance using the modified Hodge test (MHT), Rosco Neo-Sensitabs (ROSCO). Phenylalanine arginine beta-naphthylamide assay (PABN) inhibitor-based test was done on isolates in which the mechanism of resistance was not identifiable by the ROSCO. Among 105 selected isolates, carbapenemase production was noted in 100 (95%) by MHT and ROSCO showed 97 (92·4%) inhibition with dipicolinic acid signifying the production of MBL. PCR amplification was positive in 90 (86%) isolates for bla(NDM-1) and 46 (44%) isolates for bla(OXA-48). 54 (51%) isolates were positive for bla(CTX-M) and all belonged to bla(CTX-M) group 1. Isolates co produced bla(OXA-48) (31/105, 30%) and bla(CTX-M) (40/105, 38%) in combination with the carbapenemase (bla(NDM-1)) gene. Five colistin-resistant isolates were positive for bla(OXA-48). Eight isolates did not show inhibition with any of the inhibitor containing disks and found to be positive for bla(OXA-48). Isolates were tested for colistin-meropenem synergy and detection rate was higher by the checkerboard (48%) than E-test method (35%). Our study necessitates continuous surveillance to recognize the predominant machinery of resistance in a particular geographical region to formulate effective control measures. PMID:24871673

SUMMARY Objectives Limited clinical experience exists regarding the management of prosthetic joint infection (PJI) due to multidrug-resistant (MDR) Gram-negative organisms. We review three cases of carbapenem-resistant Klebsiellapneumoniae (CRKP) complicating PJI. Methods This was a retrospective study of all patients at a tertiary care institution with CRKP complicating PJI between January 2007 and December 2010. Demographic data, procedures, organisms involved, length of stay, antibiotic treatments, and outcomes were collected. Antimicrobial susceptibility testing was performed on CRKP isolates, and the mechanism of resistance was ascertained by PCR. Results This analysis demonstrated that: (1) the CRKP possessed blaKPC and were difficult to eradicate (persistent) in PJI; (2) multiple surgeries and antibiotic courses were undertaken and patients required a prolonged length of stay; (3) resistance to colistin and amikacin emerged on therapy; (4) the same strain of CRKP may be responsible for relapse of infection; (5) significant morbidity and mortality resulted. Conclusions These cases highlight the opportunistic and chronic nature of CRKP in PJIs and the need for aggressive medical and surgical treatment. Further investigations of the management of CRKP PJI and new drug therapies for infections due to MDR Gram-negative organisms are urgently needed. PMID:24813874

Over a period of 40 months, plasmid-mediated AmpC β-lactamases were detected in Tunis, Tunisia, in 78 isolates (0.59%) of Escherichia coli, Klebsiellapneumoniae, and Proteus mirabilis. In 67 isolates, only one ampC gene was detected, i.e., blaCMY-2-type (n = 33), blaACC (n = 23), blaDHA (n = 6) or blaEBC (n = 5). Multiple ampC genes were detected in 11 isolates, with the following distribution: blaMOX-2, blaFOX-3, and blaCMY-4/16 (n = 6), blaFOX-3 and blaMOX-2 (n = 3), and blaCMY-4 and blaMOX-2 (n = 2). A great variety of plasmids carrying these genes was found, independently of the species and the bla gene. If the genetic context of blaCMY-2-type is variable, that of blaMOX-2, reported in part previously, is unique and that of blaFOX-3 is unique and new. PMID:26459902

The genetic environment of two antiseptic resistance genes (qacEΔ1 and cepA) was examined in Klebsiellapneumoniae isolates obtained from the Royal Infirmary of Edinburgh between 2006 and 2008. In 4 of 34 isolates, which carried the qacEΔ1 gene, the sul1 gene was located immediately downstream. In two of these, the orf5 gene of unknown function was found immediately downstream of the sul1 gene. In one case, this was substituted by the chrA gene. The cepA gene was carried by 56 (87·5%) isolates, and the pfkA gene was found directly downstream in 45 (70·3%) isolates, and in 40 (62·5%) of these isolates, the menG gene was found directly downstream. The cpxP gene was found in 47 (73·4%) isolates upstream of the cepA, and in 35 of these isolates, the cpxR gene was identified. These latter genes are transcription regulators, and reverse transcription polymerase chain reaction (RT-PCR) revealed that their presence was associated with cepA expression. PMID:24617302

Klebsiellapneumoniae (KP) is the most common cause of neonatal sepsis in the low- and middle-income countries. Our objective was to describe the phenotypic and molecular characteristics of extended-spectrum β-lactamase (ESBL)-producer KP in neonatal care centers from Peru. We collected 176 non-duplicate consecutive KP isolates from blood isolates of neonates from eight general public hospitals of Lima, Peru. The overall rate of ESBL production was 73.3% (N = 129). The resistance rates were higher among ESBL-producer isolates when compared with the nonproducers: 85.3% versus 12.8% for gentamicin (P < 0.01), 59.7% versus 8.5% for trimethoprim-sulfamethoxazole (P < 0.01), 45.0% versus 8.5% for ciprofloxacin (P < 0.01), and 36.4% versus 12.8% for amikacin (P < 0.01). A total of 359 β-lactamase-encoding genes were detected among 129 ESBL-producer isolates; 109 isolates (84.5%) carried two or more genes. Among 37 ESBL-producer isolates randomly selected, CTX-M-15 and CTX-M-2 were the most common ESBLs detected. Most of the isolates (92%) belonged to the group KpI. Pulsed-field gel electrophoresis showed that multiple KP clones were circulating among the eight neonatal units included. PMID:26643537

Klebsiellapneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is increasingly becoming multiple drug resistant. However, the molecular pathogenesis of Kp in causing tissue injury and dysregulated host defense remains elusive, further dampening the development of novel therapeutic measures. We have previously screened a series of synthetic antimicrobial beta-sheet forming peptides and identified a peptide (IRIKIRIK; ie, IK8L) with a broad range of bactericidal activity and low cytotoxicity in vitro. Here, employing an animal model, we investigated the antibacterial effects of IK8L in acute infection and demonstrated that peritoneal injection of IK8L to mice down-regulated inflammatory cytokines, alleviated lung injury, and importantly, decreased mortality compared to sham-injected controls. In addition, a math model was used to evaluate in vivo imaging data and predict infection progression in infected live animals. Mechanistically, IK8L can kill Kp by inhibiting biofilm formation and modulating production of inflammatory cytokines through the STAT3/JAK signaling both in vitro and in vivo. Collectively, these findings reveal that IK8L may have potential for preventing or treating Kp infection. PMID:25709431

The mechanisms of killing of Klebsiellapneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing. Images PMID:8432605

Objective. To identify risk factors for the first episode of KlebsiellaPneumonia resistant to carbapenems (KPRC) infection in critically ill patients. Design, Setting, and Methods. This prospective cohort study was conducted in a 12-bed general Intensive Care Unit (ICU) in a University Hospital on ICU patients who required mechanical ventilation (MV) for >48 hours during a 12-month period. Clinical and microbiologic data were studied. Characteristics of KPRC patients were compared with those of critically ill patients who presented nonmultidrug resistant (MDR) bacterial infections or no documented infection at all. Results. Twenty-five patients presented KPRC infection, 18 presented non-MDR bacterial infection, and 39 patients presented no infection. Compared to patients without documented infection or infected by non MDR bacteria, patients with KPRC infection had received more frequently or for longer duration antibiotics against Gram-negative bacteria (carbapenems, colistin P < 0.05). Duration of colistin administration prior to KPRC isolation was independently associated with increased frequency of KPRC infection (odds ratio, 1.156 per day; 95% confidence interval, 1.010 to 1.312; P = 0.025). KPRC patients stayed longer in the ICU and received mechanical ventilation and sedation for longer periods and presented increased mortality (P < 0.05). Conclusion. KPRC infection is an emerging problem which might be more common in patients with previous use of antibiotics and especially colistin. PMID:24455733

Extended-spectrum β-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M β-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25). In some countries, CTX-M β-lactamases are the most prevalent types of ESBLs, for instance in Russia, Greece, Spain, Switzerland, Japan, Taiwan, China and Argentina. These enzymes have been identified in countries near Croatia such is Italy, Hungary and Austria. The aim of this study was to determine the prevalence and the types of CTX-M β lactamases produced by Klebsiellapneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital-based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test. PMID:26987165

Klebsiellapneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is increasingly becoming multiple drug resistant. However, the molecular pathogenesis of Kp in causing tissue injury and dysregulated host defense remains elusive, further dampening the development of novel therapeutic measures. We have previously screened a series of synthetic antimicrobial beta-sheet forming peptides and identified a peptide (IRIKIRIK; ie, IK8L) with a broad range of bactericidal activity and low cytotoxicity in vitro. Here, employing an animal model, we investigated the antibacterial effects of IK8L in acute infection and demonstrated that peritoneal injection of IK8L to mice down-regulated inflammatory cytokines, alleviated lung injury, and importantly, decreased mortality compared to sham-injected controls. In addition, a math model was used to evaluate in vivo imaging data and predict infection progression in infected live animals. Mechanistically, IK8L can kill Kp by inhibiting biofilm formation and modulating production of inflammatory cytokines through the STAT3/JAK signaling both in vitro and in vivo. Collectively, these findings reveal that IK8L may have potential for preventing or treating Kp infection. PMID:25709431

The BlrP1 protein from the enteric bacterium Klebsiellapneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FAD ox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FAD ox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FAD ox in the signalling state to the fully reduced hydroquinone form FAD redH - and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.

Circular dichroism and 1H NMR were used to investigate the interactions of a series of synthetic antimicrobial peptides (AMPs) with lipopolysaccharides (LPS) isolated from Pseudomonas aeruginosa and Klebsiellapneumoniae. Previous CD studies with AMPs containing only three Tic-Oic dipeptide units do not exhibit helical characteristics upon interacting with small unilamellar vesicles (SUVs) consisting of LPS. Increasing the number of Tic-Oic dipeptide units to six resulted in five analogues with CD spectra that exhibited helical characteristics on binding to LPS SUVs. Spectroscopic and in vitro inhibitory data suggest that there are two possible helical conformations resulting from two different AMP-LPS binding mechanisms. Mechanism one involves a helical binding conformation where the AMP binds LPS very strongly and is not efficiently